BACKGROUND: Anti-insulin antibodies (AIA) occur in diabetic dogs after insulin therapy, although their clinical significance is unclear. HYPOTHESIS: Treatment of diabetic dogs with heterologous insulin is more likely to stimulate production of AIA than is treatment with homologous insulin. ANIMALS: Diabetic dogs sampled before insulin therapy (n = 40), diabetic dogs sampled following treatment with porcine (homologous) insulin (n = 100), bovine (heterologous) lente insulin (n = 100), or bovine protamine zinc (PZI) insulin (n = 20), and nondiabetic control dogs (n = 120). METHODS: Prospective observational study. Sera were analyzed by ELISA for antibodies against porcine insulin, bovine insulin, insulin A, B, or C peptides, and control antigens; canine distemper virus (CDV) and canine thyroglobulin (TG). Canine isotype-specific antibodies were used to determine total and anti-insulin IgG1 : IgG2 ratios. RESULTS: There was no difference in CDV or TG reactivity among the groups. AIA were detected in 5 of 40 newly diagnosed (untreated) diabetic dogs. There was no significant difference in AIA (ELISA optical density reactivity) comparing control and porcine insulin-treated diabetic dogs (P > .05). Anti-insulin reactivity was most prevalent in bovine PZI insulin-treated dogs (90%; P < .01), and bovine lente insulin-treated dogs (56%; P < .01). AIA induced by treatment were enriched for the IgG1 isotype. CONCLUSIONS AND CLINICAL IMPORTANCE: This study indicates that bovine insulin is more immunogenic than porcine insulin when used for treatment of diabetic dogs.