Objective To investigate the potential role of colonic clock gene CRY1 in diarrheal irritable bowel syndrome (IBS-D). Methods An IBS-D model was constructed by mother-infant separation, acetic acid enema combined with hunger and satiety disorders, and the pups on the first day of birth were randomly divided into control group, IBS-D group and IBS-D+LL group (LL: Light:Light=12h:12h, which is 24h continuous light). The IBS-D progression was evaluated in each group, including Sucrose preference test (SPT), fecal moisture content (FMC), fecal frequency (FF) and water avoidance stress (WAS). Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of clock gene CRY1 in IBS-D mice, and the expression of melatonin receptors (MT1 and MT2) and inflammatory factors (TNF-α and IL-6) in each group of mice. Immunofluorescence and Western Blot (WB) was used to detect CRY1 expression, immunohistochemistry was used to detect melatonin synthase (Aanat), melatonin (MT) and Tryptase expression, and toluidine blue staining was used to observe colonic mast cells (MCs) in each group of mice. Results Compared with the Control group, the IBS-D group decreased SPT (P<0.05) and elevated FMC and FF (P<0.05); down-regulated the expression of CRY1, Aanat, MT, MT1 and MT2 (P<0.05); and up-regulated the expression of MCs, Tryptase, TNF-α and IL-6 (P<0.05). Compared with the IBS-D group, the IBS-D+LL group significantly decreased SPT (P<0.05) and increased FF (P<0.01); further downregulated the expression of CRY1, Aanat, MT, MT1 and MT2 (P<0.05); and further increased the expression of MCs, Tryptase, TNF-α and IL-6 (P<0.05). Conclusions Downregulation of clock gene CRY1 expression exacerbates the development of IBS-D and may be associated with reduced colonic MT expression, activation of MCs and exacerbation of low-grade inflammation in the intestinal mucosa.