Background Psoralidin (PSO), a natural phenolic coumarin, was reported to have anti-cancer activities. PSO induced reactive oxygen species (ROS) generation in cancer cells. The role of ROS in its anti-cancer effect remains unclear. Purpose This study was designed to investigate the potential roles of ROS in PSO-induced anti-cancer effect in MCF-7 breast cancer cells. Methods Effect of PSO on cancer cell proliferation was determined by MTT assay. Comet assay was used to determine DNA damage. Protein expression was detected by Western blotting. Autophagic vacuoles were detected by monodansylcadaverine (MDC) staining. ROS generation was measured by fluorescent probe. NOX4 localization was determined by immunofluorescence staining. Results PSO treatment caused proliferation inhibition in time- and dose- dependent manners, which was partially reversed by N-acetyl cysteine (NAC) and diphenyleneiodonium (DPI). PSO induced DNA damage and increased protein expression of γ-H2AX, phosphorylation of ATM, ATR, Chk1, and Chk2. PSO induced autophagy as evidenced by the accumulation of autophagic vacuoles and alterations of autophagic protein expression. PSO-induced cell death was enhanced by autophagy inhibitor chloroquine (CQ). Furthermore, PSO treatment induced ROS formation, which was reversed by NAC or DPI pretreatment. The expression of NOX4 was significantly enhanced by PSO. Both NAC and DPI could reverse PSO-induced DNA damage and autophagic responses. In addition, silencing NOX4 by siRNA inhibited PSO-induced ROS generation, DNA damage, and autophagy. Conclusions Taken together, these results showed that PSO induced DNA damage and protective autophagy mediated by ROS generation in a NOX4-dependent manner in MCF-7 cells.