Background/Aim: Melatonin, an endogenously secreted indoleamine hormone that is produced in the pineal gland, is known to possess antitumor effect via various mechanisms including induction of apoptosis and pro-oxidant effects in various cancer cells, including colorectal cancer (CRC). In our study, we hypothesized that melatonin enhances the anticancer effects via suppression of PrP C and PINK1 levels, thereby increasing superoxide production. Materials and Methods: To investigate the antitumor effects of melatonin in CRC cells, assessing its effects on mitochondrial dysfunction, production of superoxide, induction of endoplasmic reticulum stress, and cellular apoptosis were assessed. Results: Melatonin was found to decrease the expression of PrP C and PINK1, and increase superoxide accumulation in the mitochondria. In addition, PrP C -knockdown potentiated the effects of melatonin resulting further in significantly reduced expression of PINK1 and increased superoxide production in CRC. si-PRNP-transfected CRC cells treated with melatonin increased the production of intracellular superoxide and induced endoplasmic reticulum stress associated protein, and apoptosis. Conclusion: Melatonin induces mitochondria-mediated cellular apoptosis in CRC cancer cells via a PrP C -dependent pathway. PrP C knockdown combined with melatonin amplifies the effects of melatonin, suggesting a novel therapeutic strategy in targeting CRC cells.