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Background: The Müllerian duct (MD), the primordium of the female reproductive tract, is also formed in males during the early stage of development, then regresses due to the anti‐Müllerian hormone (AMH) secreted from the testes. However, the detailed diffusion pathway of AMH remains unclear. We herein investigated the mechanism by which AMH reaches the middle region of the MD using an organ culture system. Results: Injection of recombinant human AMH into the testis around the start of MD regression induced diffuse immunoreactivity in the mesonephros near the injection site. When the testis and mesonephros were cultured separately, the diameters of both cranial and middle MDs were significantly increased compared to the control. In the testis–mesonephros complex cultured by inhibiting the diffusion of AMH through the cranial region, the cranial MD diameter was significantly increased compared to the control, and there was no difference in middle MD diameter. Conclusions: These results indicate that AMH, which infiltrates from the testis through the cranial region at physiological concentrations, induces regression of the cranial MD at the start of MD regression. They also indicate that AMH infiltrating through the caudal regions induces regression of the middle MD. Key Findings: In the early stage of Müllerian duct (MD) regression, testicular anti‐Müllerian hormone (AMH) reaches the vicinity of the MD without the circulatory system.AMH infiltrates the mesonephros from the region between the testis and the mesonephros.AMH infiltrating from the testis regresses both cranial MD and middle MD. [ABSTRACT FROM AUTHOR]