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Rb is a tumor suppressor, and regulates various biological progresses, such as cell proliferation, development, metabolism and cell death. In the current study, we show that Rb knockout in 3T3 cells leads to oxidative redox state and low mitochondrial membrane potential by regulating mitochondrial activity. Our results indicate that Rb plays an important role in controlling redox homeostasis. More importantly, the functions of Rb in modulating cell proliferation, death and transformation are, at least in part, mediated by its controlling cellular redox state. In addition, our results also suggest that the cellular redox state possibly determines various biological activities, including cell survival, death and transformation, where Rb is functioning as a regulator of redox homeostasis.