Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide and has become a recognized global health problem. Therefore, the search for new anti-CRC agents or the exploration of new effective drug targets for CRC therapy is urgent. Chloroquine (CQ) is a widely-used antimalarial drug and has shown anti-proliferative effects in CRC. However, the underlying mechanisms are not well understood, particularly as the direct targets of CQ have not been identified. In this study, choline kinase alpha (CHKA) and ATP-dependent 6-phosphofructokinase, muscle type (PFKM) were identified and verified as the binding targets of CQ. CQ specifically binds to CHKA, inhibits its expression and enzymatic activity, and downregulates the downstream phosphorylation of PI3K and AKT, thereby suppressing tumor cell proliferation and inducing apoptosis. CQ also binds to PFKM and inhibits its expression and activity, thereby blocking the Warburg effect. In addition, the downregulation of CHKA can decrease the expression of PFKM and inhibit its activity, thereby blocking the Warburg effect. These observations shed new light on the antitumor mechanisms of CQ and provide new evidence for the close relationship between the PI3K/AKT signaling pathway and the Warburg effect, providing new therapeutic targets for treating CRC.