The aim of this study was to evaluate the efficacy of zinc gluconate, either associated or not to dimethyl sulfoxide (DMSO), as a contraceptive method for the canine male population. Twenty-nine sexually mature male dogs were divided into five groups: Group I (control, saline solution); Group II (zinc gluconate 13.1 mg); Group III (zinc gluconate 26.2 mg); Group IV (zinc gluconate 13.1 mg and DMSO 0.5%); Group V (zinc gluconate 26.2 mg and DMSO 0.5%). Chemical injection was directly administered into the dorsal cranial portion of each testicle once. Animals were examined for testicular size and spermogram 15 days before chemical injection, on Day 0, and then every 15 days for 6 months. Pain and sensitivity were observed in one animal of Group III at the first day after treatment. Signs of eosinophilia were observed 120 days after drug injection in Groups II and III. Dogs did not become azoospermic, but it was observed that from the second collection on, Group V sperm cell number per mm3 values were significantly lower than control group. Individual group analysis showed a significant cell motility decrease for Groups III, IV and V. Group V motility values were significantly lower than control group for collections 2, 3, 4, 5 and 8. Twelve months after chemical injection, two dogs of the control group and four from the group with more spermatic alterations (Group V) were surgically neutered and their testicles were examined through histopathological staining, revealing testicular degeneration, decreased number of germ cells, areas of atrophy, disruption of seminiferous tubule architecture, and loss of germ and Sertoli cells. The association of DMSO (0.5%) to zinc gluconate (26.2 mg) may be indicated as a contraceptive method for male dogs.