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Background: Vasectomy causes spermatozoa accumulation in the epididymis, which may cause epididymitis. Inflammation is triggered by alert molecules released following tissue stress or injury. These include uracil‐diphosphate glucose (UDP)‐glucose, which activates the pro‐inflammatory P2Y14 receptor (P2Y14), and induces immune cell recruitment. However, little is known about P2Y14 in the epididymis and its potential activation following vasectomy. Objectives: (i) To localize P2Y14 in the human excurrent duct; and (ii) to examine the effect of vasectomy on P2Y14 protein and P2RY14 mRNA content, the production of selected cytokines and chemokines, and immune cell recruitment in the epididymis. Material and methods: In situ hybridization, qRT‐PCR, western blotting, immunohistochemistry, and immunofluorescence were performed in banked human epididymis samples. Results: P2RY14 mRNA and P2Y14 protein were detected in epithelial cells in the efferent duct, epididymis and vas deferens in non‐vasectomized men. Keratin 5 (KRT5)‐positive basal cells were strongly labeled for P2Y14 in all epididymal segments. A progressive apical localization was detected in principal cells (negative for the proton pump V‐ATPase) from the corpus to the cauda. A subset of V‐ATPase‐positive clear cells also showed strong P2Y14 labeling. Vasectomy induced an increase in P2RY14 mRNA in the corpus and cauda, and stronger apical labeling in principal cells in the corpus. CXCL10 mRNA increased in the cauda and CCL2 mRNA decreased in the corpus of vasectomized versus non‐vasectomized men. No change in IL‐8 and IL‐1β mRNA was detected. Numerous CD45+ leukocytes were detected in the interstitium of the corpus and cauda following vasectomy, while only a few were seen in non‐vasectomized men. Several CD45+ leukocytes, some of which containing spermatozoa, were detected in the corpus lumen following vasectomy. Discussion and conclusion: Our study indicates that vasectomy‐induced spermatozoa congestion may lead to an inflamed‐prone local environment characterized by potential activation of P2Y14 and recruitment of immune cells in the epididymis. [ABSTRACT FROM AUTHOR]