GnRH II has important functional effects in steroid hormone-dependent tumours. Here we investigated the expression and regulation of GnRH II in prostate cancer. GnRH II protein was equally expressed in benign (73%) and malignant (78%) biopsies studied in a prostate tissue microarray (P = 0.779). There was no relationship between expression and clinical parameters in the cancer cohort. GnRH II was, however, significantly reduced in tumour biopsies following hormone ablation. This was further investigated in a prostate xenograft model where androgens increased GnRH II levels, while their withdrawal reduced it. In cell lines, we confirmed high levels of GnRH II in androgen receptor (AR)-positive LNCaP cells but low levels in AR-negative PC3 cells. In LNCaP cells, GnRH II induction by androgens was blocked by the AR inhibitor casodex, but not by cycloheximide treatment. Sequence analysis subsequently revealed a putative androgen response element in the upstream region of the GnRH II gene and direct interaction with the AR was confirmed in chromatin immunoprecipitation experiments. Finally, to test whether the effects of GnRH II were dependent on AR expression, LNCaP and PC3 cells were exposed to exogenous peptide. In both cell lines, GnRH II inhibited cell proliferation and migration, suggesting that its function is independent of AR status. These results provide evidence that GnRH II is widely expressed in prostate cancer and is an AR-regulated gene. Further studies are warranted to characterise the effects of GnRH II on prostate cancer cells and investigate its potential value as a novel therapy.