The use of GnRH antagonists is discussed as a possible non‐surgical approach to castration of male dogs. Information about histological effects on spermatogenesis is, however, missing. Therefore, we investigated the effect of a single GnRH antagonist treatment on spermatogenesis in canine testes. Sexually mature, healthy male dogs (n = 4) were treated subcutaneously with a single injection of the GnRH antagonist acyline (330 μg/kg) and surgically castrated two weeks later. Five untreated normospermic dogs served as controls. From each dog, 200 cross‐sections of approximately round tubuli seminiferi contorti were evaluated for whether there was spermatogenic arrest or undisturbed spermatogenesis. In case of full spermatogenesis being present, the different tubules were categorized in stages (Stage I‐VIII). Additionally, the area of 100 approximately round tubules from each dog was determined. The investigators were blinded to the group the dogs belong to. Histological changes in the tubular structure with a disruption of the normal cellular distribution of the testicular tissue was observed in all treated dogs with occurrence of spermatogenic arrest being significantly higher at the level of round (p = 0.0199) and elongating spermatids (p = 0.0108) compared to the control group. Percentage distribution of spermatogenic stages among seminiferous tubules differed significantly for all stages (from p = 0.0160 to p = 0.0179), except stage VIII. Furthermore, the tubular area was significantly reduced in treated compared to control dogs (p < 0.001). The histological changes in the tubular structure of the testicular tissue indicate that a single acyline treatment has a negative impact on canine spermatogenesis. (The authors thank the National Institutes of Health, USA for provision of acyline to CG.)