Objective To investigate the regulatory effect of Lizhong pills on the IL-22-MUC2/ claudin-2 signaling pathway in the colonic mucosa of spleen-deficiency diarrhea rats. Methods A spleen-deficiency diarrhea rat model was established using a 1. 2 g/ mL decoction of senna leaf combined with fatigue swimming. Model rats were randomly divided into a model, montmorillonite powder (0. 9 g/ kg), and Lizhong pill low-dose and high-dose groups (0. 81, 3. 24 g/ kg), with eight rats in each group, and eight rats were selected for a blank group. The drugs were continuously administered for 14 days. After modeling, the rats had lost weight and displayed mental fatigue, squinting, less movement, soft or loose stools, and filth around the anus, indicating that the model was successfully replicated. Serum amylase and D-xylose levels were detected by colorimetry, and colonic mucosal morphology was detected by Alcian blue and periodic acid Schiff (ABPAS) staining. A Western Blot was used to detect the expression of aquaporin 3/4 (AQP3, AQP4), and Real-time fluorescence quantitative PCR to detect the relative expression levels (2^-ΔΔCt value) of interleukin-22 (IL-22) and claudin-2 genes. An immunofluorescence method was used to detect IL-22 and claudin-2 proteins in the colon tissue. Results Compared with the blank group, the model group rats had significantly increased defecation rate, defecation grade, and diarrhea index. Epithelial cells in the colon tissue of the model group were atrophied to varying degrees; the mucosal surface structure was loose and edemic, and the goblet cells had decreased in number. The serum amylase and D-xylose contents and the expression of AQP3 and AQP4 proteins in the colon tissue were decreased, while the protein and mRNA expressions of IL-22 decreased, and those of claudin-2 were increased in the model group. Compared with the model group, the high-dose Lizhong pill group displayed significantly reduced loose stool rate, loose stool grade, and diarrhea index; significantly reduced pathological damage to the colon mucosa; increased serum amylase and D-xylose contents; increased expression of AQP3 and AQP4 proteins, increased IL-22 mRNA, and decreased claudin-2 mRNA and protein in the colon tissue, and the differences were statistically significant (P< 0. 05 or P< 0. 01). Conclusions Lizhong pills significantly improved the structure and function of the colonic mucosal barrier in spleen-deficiency diarrhea rats, and the mechanism may be related to an increase in AQP3/4 expression and regulation of the IL-22-MUC2/ claudin-2 signaling pathway.