Vitamin K2 has been shown to exert remarkable anticancer activity. However, the detailed mechanism remains unclear. Here, our study was the first to show that Vitamin K2 significantly promoted the glycolysis in bladder cancer cells by upregulating glucose consumption and lactate production, whereas inhibited TCA cycle by reducing the amounts of Acetyl-CoA. Moreover, suppression of PI3K/AKT and HIF-1α attenuated Vitamin K2-increased glucose consumption and lactate generation, indicating that Vitamin K2 promotes PI3K/AKT and HIF-1α-mediated glycolysis in bladder cancer cells. Importantly, upon glucose limitation, Vitamin K2-upregulated glycolysis markedly induced metabolic stress, along with AMPK activation and mTORC1 pathway suppression, which subsequently triggered AMPK-dependent autophagic cell death. Intriguingly, glucose supplementation profoundly abrogated AMPK activation and rescued bladder cancer cells from Vitamin K2-triggered autophagic cell death. Furthermore, both inhibition of PI3K/AKT/HIF-1α and attenuation of glycolysis significantly blocked Vitamin K2-induced AMPK activation and subsequently prevented autophagic cell death. Collectively, these findings reveal that Vitamin K2 could induce metabolic stress and trigger AMPK-dependent autophagic cell death in bladder cancer cells by PI3K/AKT/HIF-1α-mediated glycolysis promotion.