Objective:To observe the protective effect of JianpiJiedu Formula(JPJDF)on 5-FU-induced intestinal mucositis in rats through inhibiting TLRs/NF-κB signaling pathway. MethodsSixty-six SD rats were randomly divided into blank group, model group, JPJDF-Low, JPJDF-Medium and JPJDF-High groups of(0.31 g/mL, 0.62 g/mL, 1.24 g/mL)and Bifico group with 10 rats in each group. All the rats except the rats in the blank group were injected intraperitoneallywith 5-FU (50 mg/kg) once a day for 5 consecutive days. Rats in Chinese medicine treatment groups were given low, middle and high dose of JPJDF(3.1 g/kg, 6.2 g/kg, 12.4 g/kg)respectively, once a day for 7days during the chemotherapy, while those inBifico group were given Bifico (151mg /kg)daily for 7days. Diarrhea and body weight of rats were recorded daily. Seven days later, HE staining was used to observe the structure of rat small intestine. Immunohistochemistry was used to detect the expression of Occludin protein in small intestine.The serum levels of TNF-α and IL-10 were measured by ELISA, and the expressions of MyD88, p65, and TLRs in the intestine were detected by Western blot.  Results:①During the experiment, the weight of the rats in the blank group increased progressively. From the 3rd day, the weight of the rats in the model, JPJDF-Low, JPJDF-Medium, JPJDF-High and Bifico groups began to decrease. From the 5th day of the experiment, the rats in the JPJDF-High group had milder weight loss compared with those in the model group(P<0.05). Diarrhea occurred in all groups except the blank group from the 5th day, and reached the peak on the 6th day, and the incidence of diarrhea in the JPJDF-High group was lower than that in the model group on the 6th day (P<0.05).②The expression of Occludin protein in the model group decreased(P<0.01). Compared with the model group, the expression of Occludin protein in all dose groups of JPJDF increased, among which the JPJDF-High group increased most significantly(P<0.01).③The expression of serum TNF-α in the model group was higher than that in the blank group(P<0.05). Each treatment lowered its level and the TNF-α in the groups of JPJDF-Medium and JPJDF-High was lower than that in the JPJDF-Low group(P<0.05). IL-10 in the model group was lower than that in the normal group(P<0.05), and its level in the JPJDF-High group and Bifico group were higher than that in the model group(P<0.05). ④The expressions of MyD88, p65, TLR4 protein in model group was higher than those in the blank group(P<0.01), while the expressions in the JPJDF-Medium and JPJDF-High groups and Bifico group were lower than those in the model group, and theexpressions also decreased in the JPJDF-Medium and JPJDF-High groups compared with JPJDF-Low group.  Conclusion:JPJDF can inhibit TLRs/NF-κB signaling pathway and regulate inflammatory related factors, finally alleviating the 5-FU-induced intestinal mucosal inflammation in rats.