Abstract Increasing evidence from epidemiologic and pathologic studies suggests a role of macrophages in the initiation and progression of prostate cancer (PCa). During PCa development, macrophages (CD68+) are recruited into the human prostate cancer microenvironment, including inflammation-associated M1 and cancer-promoting M2 macrophages. Like other prostate cancer-associated stromal cells, macrophages express the androgen receptor (AR); however, its functionality is not known. CD68+ cells generated in vitro from peripheral white blood cells and PMA activated and IFN and LPS polarized human acute monocytic leukemia THP-1 cells also expressed AR. In these cells AR is an 80KD splice variant, lacking parts of exon 1 and exon 2. AR translocated to the nucleus upon testosterone stimulation, which was counteracted by the AR signaling inhibitor RD162. In vitro generated M1 macrophages expressed the M2 markers CD163 and CD206 upon testosterone stimulation, which was reversed by RD162. AR-ChIP sequencing suggested AR regulated Macrophage Triggering Receptor 1 (TREM1) signaling. Expression of key cytokines (e.g., CCL2, CXCL8, CCL7, IL10, IL1B) involved in TREM1 signaling was upregulated upon testosterone stimulation. Actions of these cytokines is generally associated with a protumor phenotype of macrophages. Conditioned medium from macrophages stimulated with testosterone for 8 hrs resulted in enhanced proliferation and migration of human prostate LNCap and CWRR1 cells. In conclusion, AR signaling might affect differentiation of prostate cancer-associated macrophages into cancer-promoting M2, which affects prostate cancer cell proliferation and migration. Maintaining macrophages in M1 might be a novel mechanism of action of androgen receptor inhibitors. Citation Format: Bianca Cioni, Ekatarina Nevedomskaya, Suzan Stelloo, Monique Melis, Johan van Burgsteden, Jeroen de Jong, Henk van der Poel, Jan Paul de Boer, Wilbert Zwart, Andries M. Bergman. Androgen receptor signaling affects macrophage differentiation in the human prostate cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5198.