Objective To investigate the effect of hydroxychloroquine (HCQ) on 5-fluorouracil (5-FU)-induced enteritis in mice and its mechanism. Methods Thirty C57B6/J mice were randomly divided into 0-, 1-, 3-, 5- and 7-day groups and sacrificed separately at day 0, 1, 3, 5, and 7 after intraperitoneal administration of 5-FU 200 μL/d (50 mg/kg) at day 1-3. The double-stranded DNA (dsDNA) levels in serum and small intestinal fluid were detected by dsDNA quantification kit. Severity of enteritis was evaluated by diarrhea score. HCT-116 cells were cultured in vitro and treated with 5-FU at different concentrations (0, 0.01, 0.1, 0.5, 1, 10 μmol/L) for 72 hours or 5-FU at 1 μmol/L for different time (24, 48, 72 hours). The dsDNA concentration in the cell culture supernatant of each group was detected by dsDNA quantification kit. Twenty-four C57B6/J mice were randomly divided into 4 groups: control group (intraperitoneal injection and intragastric administration of 200 μL/d saline), model group (intraperitoneal injection of 50 mg/kg 5-FU of 200 μL/d, intragastric administration of saline 200 μL/d), HCQ treatment group (intragastric administration of 60 mg/kg HCQ of 200 μL/d, starting at 1 day before the first intraperitoneal injection of 50 mg/kg 5-FU of 200 μL/d) and HCQ group (intragastric administration of 60 mg/kg HCQ solution of 200 μL/d). And they were sacrificed after 6 days. Small intestine lesions were observed by HE staining. Apoptotic cells in the small intestine were detected by TUNEL staining. The expression levels of CD11c, Toll-like receptor 9 (TLR9) and nuclear factor κB (NF-κB) in the small intestine were assessed by immunofluorescence staining. Interleukin-1β (IL-1β) levels in the serum were detected by ELISA. Mouse bone marrow-derived dendritic cells (BMDCs) were cultured in vitro and stimulated by dsDNA using LipofectamineTM 3000. The expression of TLR9 and NF-κB in BMDCs were detected by Western blotting and immunofluorescent staining, respectively. IL-1β level in the cell supernatant was detected by ELISA. Results Large amounts of apoptotic cells were observed in the small intestine of 5-FU-treated mice and the dynamic changes of dsDNA levels in the serum and small intestinal lavage fluid were consistent with that of diarrhea score. 5-FU triggered dsDNA release from HCT-116 cells in a dose- and time-dependent manner. HCQ alleviated the destruction in small intestinal epithelium and inhibited the expression levels of TLR9, NF-κB and IL-1β in the serum. The infiltration of a large number of dendritic cells in the small intestine of model mice was observed. After BMDCs were stimulated with dsDNA, the expression of TLR9, NF-κB, and IL-1β all significantly increased and HCQ significantly decreased. Conclusion HCQ alleviates 5-FU-induced enteritis in mice and inhibit TLR9 and NF-κB-dependent DNA sensing pathways and the secretion of IL-1β in dendritic cells.