Aromatase inhibitors (AIs) are the major types of drugs to treat hormone-dependent breast cancer. Although these drugs work effectively, cancer still recurs in many patients after treatment as a result of acquired resistance to the AIs. To characterize the resistant mechanisms, a set of MCF-7aro cell lines that acquired resistance to the AIs was generated. Through an "Omics" approach, we found that the resistance mechanisms of the three AIs (anastrozole, letrozole, and exemestane) differ and activation of estrogen receptor alpha (ERα) is critical for acquired AI resistance. Our results reveal that growth factor/signal transduction pathways are upregulated after ERα-dependent pathways are suppressed by AIs, and ERα can then be activated through different crosstalk mechanisms.