Bladder cancer (BCa) is one of the most common tumors, but its underlying mechanism has not been fully clarified. Our transcriptome analysis suggested a close link of Sirtuins, Peroxisome Proliferator-Activated Receptor (PPAR), cell cycle regulation, reactive oxygen species (ROS) metabolism, and Forkhead Box Class O (FOXO) signaling pathway in BCa. SIRT1 is a key member of Sirtuins, playing important roles in aging and energy metabolism, which has been reported to be involved in various metabolic diseases and tumors. We observed that SIRT1 was upregulated in BCa tissues at both mRNA and protein levels. By establishing a SIRT1-knockdown BCa cell model, our results suggested that proliferation and viability were suppressed. Moreover, migration rate was inhibited as well, possibly via reduction of epithelial-mesenchymal transition (EMT). In addition, cell cycle arrest was significantly induced, consisting with strongly decreased proteins involved (CDK2/4/6). Furthermore, ROS production was slightly reduced, accompanied by increasing of antioxidant enzymes and total/acetylated FOXO3a. Consistently with our Path-net analysis, we observed no significant alteration of apoptosis in the SIRT1-knockdown BCa cells. Taken together, our results suggested that SIRT1 deficiency in BCa cells could suppress cell viability by activating antioxidant response and inducing cell cycle arrest possibly via FOXO3a-related pathways.