Abstract: Recently, a novel melatonin MT1 receptor-mediated antiproliferative signaling mechanism involving transcriptional up-regulation of p27Kip1 due to paralleled stimulation of protein kinase A (PKA) and protein kinase C (PKC), as a result of respective dual activation of upstream Gαs and Gαq, has been reported in 22Rv1 and RWPE-1 human prostate epithelial cells. Here, we demonstrate that melatonin inhibits the proliferation of LNCaP and VCaP prostate cancer cells via activation of the same MT1 receptor-mediated antiproliferative signaling pathway. Knockdown of the expression of wild-type androgen receptor (AR) and/or structural/functional AR variants in LNCaP, VCaP, 22Rv1, and RWPE-1 cells resulted in abrogation of melatonin receptor-mediated antiproliferation, indicating that the antiproliferative signaling pathway MT1/(Gαs) PKA + (Gαq) PKC/p27Kip1 activated by melatonin in human prostate epithelial cells is AR dependent. Furthermore, melatonin was shown to decrease androgen/AR-mediated transactivation of the prostate-specific antigen promoter in the prostate epithelial cell lines. Together, our data indicate the presence of reciprocal functional interactions between MT1 receptor and AR signaling in malignant and nontumorigenic prostate epithelial cells. Notably, the dual actions of the MT1 receptor-mediated antiproliferative signaling, leading to down-regulation of activated AR signaling and up-regulation of p27Kip1, constitute the mechanistic basis for the potential use of melatonin in chemoprevention of prostate cancer, as well as in a novel therapeutic strategy, comprising a combination of melatonin repletion and androgen depletion, for the treatment of advanced or relapsed disease.