Oxidative stress (OXS) has been recently considered as one of anticancer strategies by taking advantage of higher vulnerability of cancer cells (than normal cells) to OXS. In fact, the successful outcomes using OXS have been reported in several cancer cases. A medicinal mushroom extract, PE isolated from Poria mushroom, has been shown to have anticancer/antitumor activity, although its anticancer mechanism has not been fully understood but may involve OXS. We investigated if PE might have anticancer effect on human bladder cancer cells through OXS in vitro. A dose-dependent (0-200 μg/ml of PE) study was first performed to assess cell viability using MTT assay. PE led to a significant reduction in cell viability with the IC50 (50% inhibitory concentration) of 100 μg/ml. A possible anticancer role of OXS was then assessed by lipid peroxidation (LPO) assay. The results indicated that PE indeed exerted ~2.1-fold greater OXS (than controls) on the cells. The anticancer mechanism of PE was further explored, focusing on glycolysis, metabolic signaling pathways, and apoptosis. Two glycolytic parameters, hexokinase (HK) activity and cellular ATP level, have significantly declined, suggesting the inhibition of glycolysis. Coupled with the reduced ATP level, AMP-activated protein kinase (AMPK) was activated, while protein kinase B (Akt) was inactivated and concomitantly mammalian target of rapamycin (mTOR) was inhibited. These results imply the growth cessation, followed by cell death. Western blot analysis also revealed that such cell death was more likely linked to apoptosis, indicated by the bcl-2 down-regulation and the Bax up-regulation. Therefore, PE is a natural anticancer agent with prooxidant activity exerting OXS, which leads to inhibition of glycolysis, modulations of metabolic signaling pathways, and ultimately apoptosis. It may have clinical implications in oral and/or intravesical administration for a safer and better therapeutic option for bladder cancer.