Puerarin, as a novel oncotherapeutic agent, may exert anticancer effects and inhibit the proliferation of cancer cells. To explore the effects of puerarin on human bladder cancer cells, and to elucidate the potential mechanism underlying these effects, a Cell Counting Kit-8 assay was used to examine the proliferation of T24 and EJ cells following puerarin treatment. The effects of puerarin treatment on the cell cycle were detected by flow cytometry (FCM), while puerarin-induced cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling and FCM, and the cellular ultrastructural morphological changes were observed by transmission electron microscopy. Cell invasion was examined using a Transwell assay with Matrigel. The expression levels of mechanistic target of rapamycin (mTOR), phosphorylated (p)-mTOR, p70-S6 kinase (p70S6K) and p-p70S6K proteins in the mTOR signaling pathway were then assessed by western blotting. The results demonstrated that puerarin may inhibit bladder cancer cell viability, block the cell cycle in the G0/G1 phase and induce apoptosis in bladder cancer cells. The expression levels of p-mTOR and p-p70S6K proteins were downregulated, while no change was observed in the expression levels of mTOR and p70S6K proteins when T-24 and EJ cells were treated by puerarin. In the present study, puerarin was demonstrated to inhibit the viability of human bladder cancer cells. These effects may be due to the puerarin-induced downregulation of proteins in the mTOR/p70S6K signaling pathway, and the present study may provide the experimental basis for puerarin to be considered as a promising novel anti-tumor drug for the treatment of bladder cancer.