Epidemiologic and preclinical studieshave shown that marine n‐3 polyunsaturated fatty acids (n‐3 PUFAs) elicit promising chemoprevention against breast cancer. Docosahexaenoic acid monoglyceride (MAG‐DHA), a docosahexaenoic acid sn‐1‐monoacylglycerol does not required pancreatic lipase to be absorbed, eliciting a better bioavailability when compared with other formulations such as DHA‐free fatty acid, DHA‐triglycerol, or DHA‐ethyl ester. However, the anticancer actions and underlying mechanisms of MAG‐DHA on breast cancer remain to be assessed. In this study, MAG‐DHA induced significant growth inhibition in MCF‐7 and MDA‐MB‐231 breast cancer cells in a dose‐dependent manner. MAG‐DHA treatment (80 µM) led to 83.8 and 94.3% growth inhibition between MCF‐7 and MDA‐MB‐231 cells, respectively. MAG‐DHA‐induced growth inhibition was tightly associated with apoptosis, as evidenced by increased active forms of caspase‐3, poly (ADP‐ribose) polymerase (PARP) and caspase‐12. In particular, MAG‐DHA‐induced apoptosis was triggered by oxidative stress‐mediated endoplasmic reticulum (ER) stress, as evidenced by activation of the PERK‐eIF2α pathway in ER. MAG‐DHA treatment also strongly suppressed the growth of E0771 murine breast cancer xenografts, significant differences of tumor volume were found between MAG‐DHA group (0.271 cm3) and control group (0.875 cm3) after 15 daily MAG‐DHA treatments. The in vitro antibreast cancer mechanism of MAG‐DHA was supported by the in vivo xenograft model. In addition, MAG‐DHA‐induced ER stress concomitantly triggered autophagy in these cancer cells, and the induction of autophagy suppressed its ability to induce apoptotic cell death. Our data suggested that MAG‐DHA as dietary supplement, in combination with autophagy inhibitors may be a useful therapeutic strategy in treating breast cancer.