Objective: To investigate the photodynamic killing effect mediated by Cu-Cy under X-ray activation on human colon cancer SW620 cells in vitro and the mechanism. Methods: SW620 cells were exposed to different concentrations of Cu-Cy (0–100 mg/L) in combination with or without low dose of X-ray radiation, and then, the cell viability was examined by CCK8 assay and light microscopic observation, mitochondrial membrane potential was detected by JC-1 dye, apoptosis was measured by Annexin V/PI double-staining, the expressions of apoptosis-associated protein Bax and Bcl-2, and autophagy-associated protein LC3B and P62 were determined by Western blot analysis, and cell ultrastructure was observed by transmission electron microscopy. Results: In SW620 cells compared with blank control SW620 cells (without Cu-Cy treatment and X-ray radiation), the cell viability was significantly reduced with certain concentration-dependent manner (part P<0.05), and meanwhile, the ratio of cells with decreased mitochondrial membrane potential and the apoptotic rate were significantly increased, the expression levels of Bax and LC3B-II were significantly increased while the expression levels of Bcl-2 and P62 were significantly decreased (all P<0.05), and the intracellular autophagosomes were remarkably increased; all above indexes showed no significant change in those cells that underwent X-ray radiation alone or Cu-Cy treatment alone. Conclusion: The photodynamic action mediated by Cu-Cy under X-ray activation can effectively inhibit the growth and viability of human colon cancer SW620 cells in vitro, and the mechanism may possibly be associated with its inducing apoptosis and autophagy.