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Butein is a flavonoid isolated from various medicinal plants. It is known to have different biological activities including anti‐inflammation, anti‐adipogenesis, and anti‐angiogenesis. In the study, we demonstrated the anti‐proliferative effect of butein in human osteosarcoma U‐2 OS cells. Our data showed that butein significantly suppressed the viability and colony formation ability of U‐2 OS cells. Further experiments revealed butein exposure resulted in a cell cycle arrest at S and G2/M phase in U‐2 OS cells. Importantly, we found that butein activated the tumor suppressor p53, and trigged a p53‐dependent senescence in U‐2 OS cells. Knockdown of p53 suppressed the senescence and rescued the viability in butein‐treated U‐2 OS cells. Furthermore, we observed that butein exposure significantly enhanced reactive oxygen species (ROS) levels in U‐2 OS cells. Co‐administration of the ROS inhibitor NAC largely abolished the up‐regulated p53 protein level, and rescued the suppressed viability and colony formation ability in butein‐exposed U‐2 OS cells. Taken together, our data proposed the increased ROS by butein exposure activated p53, and the activated p53 was involved in the anti‐proliferative effect of butein via inducing senescence in U‐2 OS cells. This report suggests that butein is a promising candidate for cancer therapy against osteosarcoma. [ABSTRACT FROM AUTHOR]