Background: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA positron emission tomography/computed tomography has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear. Methods: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected from patients with prostate cancer via robotic surgery in 2019 at Ehime University. In vitro, we prepared conditioned medium derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the conditioned medium. We assessed angiogenic activities by treating HUVECs with LNCaP-derived conditioned medium using a tube formation assay that mimics angiogenesis. Results: Immunohistochemical positivity of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in four of 33 prostate cancer patients (12.1%). We also found that the 10,000 ´ g pellet fraction of the LNCaP-derived conditioned medium containing PSMA-positive membranes, including microvesicles, transformed HUVECs from “PSMA-negative” to “PSMA-positive.” Mass spectrometry revealed that the fraction contained an important growth factor. Furthermore, treating HUVECs with the 10,000 ´ g pellet fraction of the LNCaP-derived conditioned medium significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner. Conclusions: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which were found to enhance tumor angiogenesis in prostate cancer tissues. The endocytic process related to these PSMA-positive microvesicles in normal endothelial cells might be an attractive target to develop novel anti-angiogenic drugs, which could inhibit the transformation of normal endothelial cells into tumor endothelial cells (Watanabe et al., The Prostate. 2021. in press).