Objective To explore the effects of various modeling times on the degree of colonic injury in mice subjected to water immersion restraint stress (WIRS) and compare the degree of inflammatory injury and the composition of intestinal flora between WIRS and control mice. Methods Thirty-four Kunming mice were used in this study. Eighteen mice were randomized into control, WIRS4 h, 12 h, 24 h, 36 h, and 48 h groups. After modeling, HE staining was performed to observe the degree of colonic injury at various modeling times and evaluate the modeling conditions. Sixteen mice were randomized into control and WIRS24 h groups. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ ( IFN-γ) contents were measured by enzyme-linked immunosorbent assays at the end of modeling. The composition of colonic contents was assessed by 16S rRNA gene sequencing. Results All WIRS groups exhibited various degrees of diffuse bleeding and reddening in the colonic lumen. HE staining showed inflammatory cell infiltration in WIRS mice. Colonic injury of the WIRS24 h group was obvious and the survival rate was high. TNF-α, IL-6, and IFN-γ levels were significantly increased in the WIRS24 h group (P<0. 0001). 16S rRNA sequencing showed intestinal flora disorder in the WIRS24 h group. Compared with the control group, the abundance of Campylobacter, Deferribacterota, Helicobacter, Bacteroides, Roseburia, norank_f__Ruminococcaceae, and unclassified_f__Oscillospiraceae was significantly increased (P<0. 05 or P<0. 01), and that of Actinobacteriota, Desulfobacterota, norank _f __norank _o __Clostridia _UCG-014, and Odoribacter was significantly decreased (P<0. 05 or P<0. 01). Conclusions The WIRS model causes histopathological damage to the colon in mice and significant alterations in the composition of intestinal flora.