Despite extensive research, the genes/proteins and biological pathways responsible for the physiological effects of estrogen remain elusive. In this study, we employed a proteomic method to determine the effect of estrogen on global protein expression in breast cancer MCF7 cells. The expression of 77 cytoplasmic, 74 nuclear, and 81 membrane proteins was significantly altered by 17-β-estradiol (E2). Protein enrichment analyses of the quantified proteins revealed that E2 stimulated cell division primarily by promoting the G1 to S phase transition and advancing the G2/M checkpoint. Functional annotation analyses of the E2-regulated proteins indicated that these proteins promoted cell division primarily through enhancing protein nuclear import, protein translation, reducing mRNA degradation, and increasing protein folding. Interestingly, many of the E2-upregulated proteins contained the HEAT, KH, and RRM domains. The effect of E2 on cell survival was complex, as it could simultaneously enhance and inhibit apoptosis. E2 enhanced apoptosis by promoting cytochrome c release from mitochondria and inhibited apoptosis by activating the PI3K/AKT/mTOR signaling pathway. Treatment of MCF7 cells with E2 and the PI3K inhibitor Ly294002 significantly enhanced apoptosis compared to the cells treated with E2 alone. Our results suggest that combining estrogen with a PI3K inhibitor could be a promising strategy for treating ERα-positive breast cancer.