Dogs may be occasionally infected by smooth strains of Brucella spp. The infection is usually associated with the ingestion of contaminated material from parturition or abortion, or other tissues from infected farm animals, particularly cattle and pigs. A 6-year-old, male mixed breed dog from a rural area was admitted at a veterinary clinic for clinical examination. The dog had bilateral perineal hernia with dysuria and dyschezia, as well as small firm testicles with scrotal adhesions. Serological tests, including buffered plate antigen, serum agglutination test, and 2-mercaptoethanol test, were positive for smooth Brucella spp. strains, whereas a rapid slide agglutination test was negative for B. canis. Blood and prostate tissue samples yielded no bacterial isolates. Histopathology demonstrated interstitial lymphoplasmacytic and histiocytic infiltration of the prostate gland, with fibrosis and occasional disruption of glandular architecture. Immunohistochemistry demonstrated abundant Brucella spp. antigens in the cytoplasm of macrophages. This report supports the notion that not only B. canis, but also smooth Brucella spp. must be considered in the differential diagnosis of prostatitis in dogs.