**Background:** Dietary fiber and non-fibrous carbohydrates play critical roles in intestinal health by modulating gut microbiota and immune function. Neutral detergent fiber (NDF) is fermented by gut microbes into volatile fatty acids (VFAs) that support intestinal barrier integrity, while non-fibrous carbohydrates (NFC) provide readily available energy. An unbalanced NFC/NDF ratio can disrupt gut microbial balance and impair growth, but limited data exist for monogastric animals. This study investigated the effects of varying dietary NFC/NDF ratios on gut microbiota, metabolites, and intestinal immunity in rabbits.
**Methods:** A total of 135 weaned New Zealand White rabbits (45.1 ± 0.7 days old, mean weight 1.08 ± 0.07 kg) were randomly assigned to five groups (27 replicates per group, one rabbit per replicate) and fed diets with NFC/NDF ratios of 0.7 (T1), 1.0 (T2), 1.3 (T3), 1.6 (T4), and 1.9 (T5) for 28 days. Growth performance (body weight, average daily gain [ADG], average daily feed intake [ADFI], feed conversion ratio [F/G]), diarrhea rate, and mortality were recorded. Blood samples were analyzed for white blood cell (WBC), neutrophil, lymphocyte, and monocyte counts. Cecal tissue mRNA expression of IL-1β, TNF-α, Claudin-1, Occludin, and ZO-1 was measured by RT-qPCR. Cecal pH, VFAs (acetate, propionate, butyrate), and NH3-N were quantified. Cecal microbiota was analyzed via 16S rRNA gene sequencing. Statistical analysis used one-way ANOVA with linear and quadratic polynomial contrasts; P < 0.05 was considered significant.
**Key Results:** Growth performance was significantly affected by NFC/NDF ratio. BW and ADG were highest in T3 (2563.38 g, 55.53 g/day) and lowest in T5 (2076.06 g, 38.63 g/day; P < 0.001 for both linear and quadratic trends). ADFI was significantly higher in T1–T3 (193.79, 179.31, 181.49 g/day) than T4 and T5 (157.04, 154.59 g/day; P < 0.001). F/G ratio was highest in T5 (4.25) vs. other groups (3.24–3.57; P = 0.002). Diarrhea and mortality rates were 0% in T1–T3 but reached 18.52% and 7.41% in T4, and 59.26% and 14.81% in T5, respectively. Blood WBC and neutrophil counts were significantly elevated in T5 vs. T1–T3 (P < 0.05). Pro-inflammatory cytokine expression (IL-1β, TNF-α) was significantly higher in T4 and T5 vs. T1–T3 (P < 0.05). Claudin-1 and Occludin expression was highest in T2 and T3 (P < 0.05); ZO-1 was highest in T2 (P < 0.05). Cecal pH decreased linearly (P_lin = 0.032) and quadratically (P_quad = 0.005) with increasing NFC/NDF, with T5 lowest (6.11) vs. T2 highest (6.52). Total VFA was highest in T5 (44.39 mmol/L) vs. T1 and T2 (40.32, 39.45 mmol/L; P = 0.010). Acetate decreased linearly (P_lin = 0.005) and quadratically (P_quad = 0.014), with T1 and T2 highest (25.70, 26.36 mmol/L) and T5 lowest (21.90 mmol/L). Propionate increased quadratically (P_quad < 0.001), with T4 and T5 (11.02, 11.54 mmol/L) significantly higher than T1–T3 (8.94, 9.27, 8.91 mmol/L). Butyrate and NH3-N showed no significant differences. Microbiota analysis revealed that Shannon diversity was lower in T5 vs. T2 and T3 (P < 0.05). At the phylum level, Firmicutes relative abundance was lowest in T5 (50.89%) vs. T2 (69.07%; P < 0.01). Bacteroidetes increased linearly (P_lin = 0.011) and quadratically (P_quad = 0.014), with T4 and T5 (26.36%, 26.13%) higher than T2 (15.14%). Verrucomicrobia was highest in T5 (14.44%; P < 0.001). Proteobacteria increased linearly and quadratically (both P < 0.001), with T5 highest (0.93%). At the genus level, beneficial Ruminococcaceae_NK4A214_group, Ruminococcaceae_UCG-014, and Ruminococcaceae_UCG-013 were higher in T2 and T3 vs. T4 and T5 (P < 0.05). Coriobacteriaceae_UCG-002 was highest in T2 (4.24%; P < 0.001). Muribaculaceae_unclassified and Akkermansia were highest in T5 (23.40%, 14.44%; P < 0.001). LEfSe analysis identified beneficial Clostridiales, Ruminococcaceae, and Coriobacteriaceae as enriched in T2, while potentially harmful Rickettsiales was enriched in T5. Correlation analysis showed Ruminococcaceae genera positively correlated with acetate and negatively with propionate (P < 0.01). Lachnospiraceae_NK4A136_group was positively correlated with butyrate (P < 0.05). Muribaculaceae_unclassified and Akkermansia were negatively correlated with acetate and butyrate (P < 0.05).
**Clinical Implications:** This study demonstrates that dietary NFC/NDF ratio is a critical determinant of intestinal health in rabbits. An NFC/NDF ratio of 1.0–1.3 optimizes growth performance, reduces diarrhea and mortality, enhances beneficial gut microbiota (Firmicutes, Ruminococcaceae, Lachnospiraceae), and promotes a favorable VFA profile (higher acetate, lower propionate) while suppressing pro-inflammatory cytokine expression and maintaining intestinal barrier integrity. Higher ratios (1.6–1.9) are associated with gut dysbiosis, increased Proteobacteria, elevated inflammation, and poorer outcomes. These findings provide a scientific basis for formulating rabbit diets and may inform nutritional strategies for other monogastric animals. The study is limited by its short duration (28 days) and the use of a single animal model; further research is needed to confirm these effects in other species and to elucidate the molecular mechanisms linking specific microbial taxa to immune modulation.