We have previously demonstrated that the p44/42 MAPK pathway is one pathway involved in homeobox (HOX) A1-stimulated oncogenesis. However, inhibition of MAPK kinase 1 does not completely prevent HOXA1-stimulated oncogenic transformation, suggesting the involvement of additional signal transduction pathways. Here, we report that forced expression of HOXA1 in immortalized human mammary epithelial cells significantly increased levels of signal transducer and activator of transcription (STAT) 3, 5A, and 5B mRNA by transcriptional up-regulation. The protein levels of STAT3 and 5B, but not STAT5A, and protein phosphorylation levels of STAT3 and 5B were significantly increased by forced expression of HOXA1. Forced expression of STAT3 or STAT5B was sufficient to transform oncogenically an immortalized human mammary epithelial cell line. Accordingly, inhibition of STAT3 or STAT5B activity with dominant negative STAT3 or STAT5B abrogated the ability of HOXA1 to stimulate cell proliferation, survival, oncogenic transformation, and generation of large disorganized multiacinar structures in three-dimensional culture. These results suggest that HOXA1 partially mediates oncogenic transformation of the immortalized human mammary epithelial cell through modulation of the STAT3 and STAT5B pathways.