Objective To study the protective effect of Paeonol on intestinal mucosal injury induced by 5-FU in mice. Methods Mice were randomly divided into control group (CON, 12 mice), 5-FU injection group (5-FU, 12 mice), paeonol 25mg/kg group (5-FU+25mg/kg Pae, 13 mice), paeonol 100mg/kg group (5-FU+100mg/kg Pae, 13 mice). The mice intestinal mucosal injury model was established by intraperitoneal injection of 5-FU, and the effects of paeonol on the weight of mice after 5-FU injection were studied. The morphological effects of paeonol on the small intestinal villus injury were observed by H&E staining, and the apoptosis of small intestinal adenocytes was studied by immunohistochemical experiment. Finally, the effect of paeonol on 5-FU antitumor activity was observed by tumorigenicity assay. Results After 9 days of 5-FU injection, the weight of mice decreased significantly, while paeonol could significantly restore the weight of mice. Paeonol can significantly reduce the degree of small intestinal edema or bleeding, and increase the number of small intestinal glands. Immunohistochemical results showed that paeonol could significantly reduce the apoptosis of small intestinal gland cells. Finally, we found that paeonol had certain anti-tumor effects and might enhance the anti-tumor activity of 5-FU. Conclusion Paeonol can reduce the effect of 5-FU on mucosal injury and enhance the pit tumor activity of mice by inhibiting the apoptosis of small adenocarcinoma cells.