Previous studies have verified the protective role of n3 polyunsaturated fatty acids (PUFAs) in cancer. Fish brain lipids are rich in n3 PUFAs. However, the action and mechanism of their potent anticancer activities remain unclear. In this study, polar lipids (PLs) and neutral lipids (NLs) were isolated from the total lipids (TLs) of the silver carp brain, and the anticancer effects of the lipid fractions (LFs) were examined in a human breast cancer cell line (MCF-7). The LFs effectively inhibited the cell proliferation of MCF-7 cells in a time- and concentration-dependent manner by cell cycle arrest at the S stage and induction of apoptosis. Further analyses showed that the apoptotic effect of the LFs on MCF-7 cells was associated with the accumulation of elevated levels of reactive oxygen species and the loss of mitochondrial membrane potential. Among the TLs, PLs were more effective at causing breast cancer cell death than NLs. Our study showed that the LFs from silver carp brains may aid the prevention and treatment of human breast cancer.