The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA-mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative ?-interferon activation sequence (GAS) element at -434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the -434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5'-flanking region containing the -434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.