Objective To explore the influence of cucurbitacin E (CuE) on autophagy in human bladder cancer cell line T24 and further study its impacts on cell proliferation. Methods MTT assay was used to determine the proliferation inhibition capacity of CuE on T24 and western blot to check the impacts of CuE treatment on the expression of classic autophagy markers LC3A/B and p62. LC3 turnover assay and GFP-RFP-LC3 fluorescent assay were performed to determine autophagy flux. Western-blot was used to check the autophagy inhibition ability of 3-MA on CuE treatment and MTT assay and cell counting assay were used to check the influence of CuE-induced autophagy on cell proliferation with/without autophagy inhibition. Results CuE inhibited the proliferation of T24 and the IC50 in 24 h was about 0.75 μmol/L. CuE treatment increased the expression of LC3A/B Ⅱ and LC3A/B Ⅱ/Ⅰ ratio (P < 0.05) , but decreased the expression of p62 (P < 0.05) , indicating the induction of autophagy. Autophagy flux was induced because of positive LC3 turnover assay and the increase of yellow and red dots in GFP-RFP-LC3 fluorescent assay (P < 0.05). CuE-induced autophagy was inhibited by 3-MA (P < 0.05). With autophagy inhibition, CuE's proliferation suppression ability on T24 was attenuated (P <0.05). Conclusion CuE induces autophagy in bladder cancer cell line T24 and the induced autophagy positively contributes to the inhibitation of cell proliferation.