Background The aim of this study was to determine the effect of dietary inclusion of silkworm pupae meal (SPM) on nutrient digestibility, nitrogen utilization, gastrointestinal physiology and blood biochemical parameters in rabbits. Thirty Termond White rabbits were divided into three groups: SBM – fed a diet containing 10% soybean meal (SBM), SPM5 – fed a diet containing 5% SBM and 5% SPM, and SPM10 – fed a diet containing 10% SPM. Results Nutrient digestibility and nitrogen retention decreased with increasing SPM inclusion levels in rabbit diets. The dietary inclusion of SPM caused a significant increase in the stomach pH. Group SPM10 rabbits were characterized by the highest cecal tissue and digesta weights. The lowest cecal pH was noted in group SPM5. The relative weights of colonic tissue and digesta tended to increase with increasing levels of SPM. The total and intracellular activity of bacterial α-galactosidase decreased significantly in both SPM groups. The replacement of SBM with SPM led to a decrease in the activity of bacterial β-glucuronidase in the cecal digesta. The intracellular activity of bacterial α-arabinofuranosidase increased, and its release rate decreased in the cecum of rabbits in SPM groups. The extracellular activity of bacterial β-xylosidase in the cecal digesta tended to decrease in group SPM10. The highest extracellular and intracellular activity of bacterial β-cellobiosidase in the cecal digesta was noted in the SPM5 treatment. The lowest and the highest activity of bacterial N-acetyl-β-D-glucosaminidase (NAGase) was observed in groups SBM and SPM10. The SPM10 treatment contributed to a decrease in the cecal concentrations of butyric, iso-valeric and valeric acids. The lowest total concentration of putrefactive short-chain fatty acids (PSCFAs) was observed in group SPM10. The cecal concentration of propionic acid tended to increase in group SPM5, whereas the cecal concentration of iso-butyric acid tended to decrease in group SPM10. The colonic concentration of iso-valeric acid was lowest in group SPM5. SPM treatments resulted in a significant increase in plasma albumin concentration. Plasma urea concentration was significantly higher in group SPM10 than in SBM and SPM5. Conclusions The results of this study suggest that rabbit diets can be supplemented with SPM at up to 5%.