Background:High-protein diets can increase the colonic health risks. A moderate reduction of dietary crude-protein (CP) level can improve the colonic bacterial community and mucosal immunity of pigs. However, greatly reducing the dietary CP level, even supplemented with all amino acids (AAs), detrimentally affects the colonic health, which may be due to the lack of protein-derived peptides. Therefore, this study evaluated the effects of supplementation of casein hydrolysate (peptide source) in low-protein (LP) diets, in comparison with AAs supplementation, on the colonicmicrobiota, microbial metabolites and mucosal immunity in pigs, aiming to determine whether a supplementation of casein hydrolysate can improve colonic health under very LP level. Twenty-one pigs (initial BW 19.90 ± 1.00 kg, 63 ± 1 days of age) were assigned to three groups and fed with control diet (16％CP), LP diets (13％CP) supplemented with free AAs (LPA) or casein hydrolysate (LPC) for 4 weeks. Results:Compared with control diet, LPA and LPC diet decreased the relative abundance of Streptococcus and Escherichia coli, and LPC diet further decreased the relative abundance of Proteobacteria. LPC diet also increased the relative abundance of Lactobacillus reuteri. Both LP diets decreased concentrations of ammonia and cadaverine, and LPC diet also reduced concentrations of putrescine, phenol and indole. Moreover, LPC diet increased total short-chain fatty acid concentration. In comparison with control diet, both LP diets decreased protein expressions of Toll-like receptor-4, nuclear factor-κB, interleukin-1β and tumor necrosis factor-α, and LPC diet further decreased protein expressions of nucleotide-binding oligomerization domain protein-1 and interferon-γ. LPC diet also increased protein expressions of G-protein coupled receptor-43, interleukin-4, transforming growth factor-β, immunoglobulin A and mucin-4, which are indicators for mucosal defense activity. Conclusions: The results showed that supplementing casein hydrolysate showed beneficial effects on the colonic microbiota and mucosal immunity and barrier function in comparison with supplementing free AAs in LP diets. These findings may provide new framework for future nutritional interventions for colon health in pigs.