The molecular mechanisms underlying progression of prostate cancer to the hormone-independent state are poorly understood. Signal transducer and activator of transcription 5a and 5b (Stat5a/b) is critical for the viability of human prostate cancer cells. We have previously shown that Stat5a/b is constitutively active in high-grade human prostate cancer, but not in normal prostate epithelium. Furthermore, activation of Stat5a/b in primary human prostate cancer predicted early disease recurrence. We show here that transcription factor Stat5a/b is active in 95% of clinical hormone-refractory human prostate cancers. We show for the first time that Stat5a/b synergizes with androgen receptor (AR) in prostate cancer cells. Specifically, active Stat5a/b increases transcriptional activity of AR, and AR, in turn, increases transcriptional activity of Stat5a/b. Liganded AR and active Stat5a/b physically interact in prostate cancer cells and, importantly, enhance nuclear localization of each other. The work presented here provides the first evidence of synergy between AR and the prolactin signaling protein Stat5a/b in human prostate cancer cells.