Background: Endocrine disruptors, including xenoestrogens, are environmental contaminants that can mimic or interfere with estrogen signaling. Exposure to these compounds has been associated with male infertility, in which up to 50% of cases remain idiopathic. Estrogen plays a complex role in immune regulation by enhancing immune responses while suppressing inflammatory pathways. An exacerbated immune response may contribute to male infertility by impairing sperm development and storage, highlighting the importance of understanding estrogen action in the male reproductive tract immunity. Here, we investigated how estrogen modulates mononuclear phagocyte (MPs) responses in the testis and epididymis during bacterial- and viral-like epididymitis. Methods: Male mice received a subcutaneous osmotic pump (Alzet D1007; flow rate = 0.48 µL h⁻¹) delivering either 1.4 µg/day of 17β-estradiol (E2) or 2-hydroxypropyl-β-cyclodextrin (vehicle). Five days after implantation, mice received an intravasal injection of saline, LPS (20 µg; TLR4 agonist), or Poly I:C (100 µg; TLR3 agonist) and were euthanized 48 h later. Luminal-reaching projections of F4/80⁺ MPs and V-ATPase immunofluorescence were evaluated in the epididymis, whereas CD68, Ki67, and PNA immunofluorescence were assessed in the testis. Results: Either in saline, LPS, or Poly I:C intravasal injection, E2 supplementation increased luminal-reaching projections of F4/80⁺ MPs in the initial segment. E2 also increased F4/80⁺ MPs in the epithelial and interstitial compartments of the epididymal cauda. Enhanced apical V-ATPase immunostaining was detected in clear cells from E2-treated mice. In testis, CD68 immunostaining increased following saline, LPS or Poly I:C intravasal injections as well as E2 supplementation whereas PNA/Ki67 immunofluorescence showed apparently intact seminiferous epithelium. Conclusion: During bacterial- and viral-like epididymitis, testicular and epididymal MPs, alongside epididymal clear cells, respond to E2. These findings suggest that E2 could modulate immune and epithelial cell responses, underscoring a possible pathway through which environmental xenoestrogens could impact immune responses in the male reproductive tract.