ARTICLE ABSTRACTPurpose: Although a previous study reported nerve ending–derived acetylcholine promoted prostate cancer invasion and metastasis by regulating the microenvironment of cancer cells, the present study aims to determine whether there is autocrine cholinergic signaling in prostate epithelial cells that promotes prostate cancer growth and castration resistance.Experimental design: In this study, IHC was performed to detect protein expression in mouse prostate tissue sections and human prostate cancer tissue sections. Subcutaneously and orthotopically xenografted tumor models were established to evaluate the functions of autocrine cholinergic signaling in regulating prostate cancer growth and castration resistance. Western blotting analysis was performed to assess the autocrine cholinergic signaling–induced signaling pathway.Results: We found the expression of choline acetyltransferase (ChAT), the secretion of acetylcholine and the expression of CHRM3 in prostate epithelial cells, supporting the presence of autocrine cholinergic signaling in the prostate epithelium. In addition, we found that CHRM3 was upregulated in clinical prostate cancer tissues compared with adjacent non-cancer tissues. Overexpression of CHRM3 or activation of CHRM3 by carbachol promoted cell proliferation, migration, and castration resistance. On the contrary, blockading CHRM3 by shRNA or treatment with darifenacin inhibited prostate cancer growth and castration resistance both in vitro and in vivo. Furthermore, we found that autocrine cholinergic signaling caused calmodulin/calmodulin–dependent protein kinase kinase (CaM/CaMKK)–mediated phosphorylation of Akt.Conclusions: These findings suggest that blockade of CHRM3 may represent a novel adjuvant therapy for castration-resistant prostate cancer. Clin Cancer Res; 21(20); 4676–85. ©2015 AACR.