Background Mitophagy is a type of selective autophagy for dysfunctional mitochondria and plays a key role in tumorigenesis and cancer progression. However, whether mitophagy plays a role in colon cancer remains unclear. Cirsiliol is a natural product and has been found to exert anti-cancer effects in multiple tumors. The effects of cirsiliol in the tumorigenesis and progression of colon cancer remain unknown. Methods CCK8 assay, plate cloning assay, and cell scratch assay were performed to determine cell viability, colony formation, and wound healing abilities of HCT116 and SW480 cells. JC-1 staining, H2DCFDA staining, and Mito-Tracker Red staining were carried out to evaluate mitochondrial membrane potential (Delta psi m), intracellular reactive oxygen species (ROS) level, and mitochondrial morphology. Molecular docking technology was utilized to predict interaction of cirsiliol and signal transducer and activator of transcription 3 (STAT3). Immunofluorescence staining was used to measure nuclear translocation of STAT3. The protein levels of phosphorylated STAT3 (Y705), total STAT3, and mitophagy proteins were detected by western blot. Results In this study, we first found that cirsiliol inhibited cell viability, colony formation, and wound healing abilities of HCT116 and SW480 colon cancer cells. Moreover, cirsiliol suppressed Delta psi m, increased ROS production, and disrupted mitochondrial morphology via inhibiting the levels of mitophagy proteins including PINK1, Parkin, BNIP3, and FUNDC1. Application of mitophagy activator improved the levels of mitophagy-related proteins, and ameliorated Delta psi m and ROS levels. According to the result of molecular docking, we found that cirsiliol potentially bound to the SH2 domain of STAT3, the key domain for the functional activation of STAT3. Moreover, it was found that cirsiliol inhibited constitutive and IL-6-induced STAT3 phosphorylation and nuclear translocation by western blot and immunofluorescence analysis. Comparing with cirsiliol group, we found that overexpression of STAT3 restored the expressions of mitophagy proteins. Conclusions Cirsiliol targets STAT3 to inhibit colon cancer cell proliferation by regulating mitophagy.