Breast cancer is a common malignancy threatening women's health around the world. Despite improved treatments for different subtypes of breast tumors that have been put forward, there still exists a poor therapeutic response and prognosis. Magnetic fields, as a non-invasive therapy, have shown anti-tumor effects in vitro and in vivo; however, the detailed mechanisms involved are still not clear. In this study, we found that in exposure to low-frequency magnetic fields (LF-MFs) with an intensity of 1 mT and frequencies of 50, 125, 200, and 275 Hz, separately, the proliferation of breast cancer cells was inhibited and LF-MF with 200 Hz reached the optimum inhibition effect, on exposure time-dependently. Notably, we found that exposure to LF-MF led to MCF-7 and ZR-75-1 cell apoptosis and cell cycle arrest. Moreover, we also discovered that LF-MF effectively increased the level of reactive oxygen species (ROS), suppressed the PI3K/AKT signaling pathway, and activated glycogen synthase kinase-3β (GSK-3β). We demonstrated that the GSK3β activity contributed to LF-MF-induced cell proliferation inhibition and apoptosis, while the underlying mechanism was associated with the inhibition of PI3K/AKT through increasing the intracellular ROS accumulation. These results indicate that LF-MF with a specific frequency may be an attractive therapy to treat breast cancers.