FTY720, also known as fingolimod, is a widely used immunomodulator in multiple sclerosis and multiple organ transplantation. It is also an important protein phosphatase 2A (PP2A) activator. Based on this, a number of studies have recently demonstrated the cytotoxic effect of FTY720 in various cancers. Yet in colorectal cancer (CRC), the underlying mechanisms of FTY720 cytotoxicity remain less clear, especially the relationship between a drug and autophagy. We demonstrate here for the first time that FTY720 promotes the appearance of autophagic hallmarks such as autophagosome formation and light chain 3 (LC3)-II accumulation, indicating the participation of autophagy in FTY720 cytotoxicity on CRC. Moreover, inhibition of autophagy using 3-methyladenine (3-MA), a specific inhibitor of autophagy, enhanced FTY720 cytotoxicity, indicating the protective role of autophagy against the drug's own cytotoxic effect. The protective autophagy was likely affected by cancerous inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor that is closely related with poor prognosis and drug resistance. Consequently, our data not only demonstrate a new mechanism underlying the cytotoxic effect of FTY720 in CRC, but also a new strategy for CRC treatment, especially in cases resistant to conventional chemotherapies because of high CIP2A levels.