Constipation is the most common gastrointestinal complaint all over the world, and it is a risk factor of colorectal cancer. In this study, the protective of Quercetin against loperamide‐induced constipation and its potential mechanism in a rat model were investigated. Results showed that Quercetin at 25 mg/kg and 50 mg/kg could significantly (p < .05) increase the intestinal transit rate, motilin, gastrin, substance P levels, and concentration of short‐chain fatty acids (SCFAs), reduce the somatostatin levels, and improve the gastrointestinal peristalsis of rats. In addition, the expression levels of enteric nerve‐related factors, glial cell line‐derived neurotrophic factor (GDNF), transient receptor potential vanilloid 1 (TRPV1), nitric oxide synthase (NOS), c‐Kit, stem cell factor (SCF), and aquaporin 3 (AQP3) were examined by RT‐qPCR and/or Western blot analysis. The results suggest that Quercetin relieves loperamide‐induced constipation by increasing the levels of interstitial cells of Cajal markers (c‐Kit and SCF), as well as AQP3. In conclusion, the present study suggested that Quercetin exerted a protective effect against loperamide‐induced constipation, which may be associated with its role in regulation of multiple signal pathways.