Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide. More than 30% of CRC patients will experience treatment failure and tumor recurrence after standard-of-care treatment. Therefore, it is important to discover new therapeutic regimens for treating CRC. Repurposing existing clinically used drugs into new anticancer agents represents a feasible way and has become increasingly popular. In this study, the aim is to investigate the anticancer effect of sertraline on CRC and to elucidate its underlying mechanism. The data showed that sertraline exhibited a potent anticancer effect against CRC in vitro and in vivo. Sertraline inhibited Akt- and STAT3-mediated cell proliferation but do not affect several programmed cell deaths in CRC, such as apoptosis, pyroptosis, ferroptosis, and mitophagy. Meanwhile, sertraline induced autophagosome accumulation but blocked autophagic flux in CRC cells. Further investigations reveal that sertraline impeded late autophagic flux at the stage of autolysosomal degradation rather than autophagosome-lysosomal fusion in CRC. Furthermore, it is also demonstrated that sertraline synergistically sensitized chemotherapeutic agents against CRC. Overall, the study reveals the great potential of sertraline as a novel therapeutic candidate for CRC, which is worthy of further development in the future.