Leptospirosis is one of the important zoonotic bacterial diseases with a worldwide distribution that is often subclinical. We aimed to investigate the oxidant/antioxidant balance and acute‐phase response in naturally infected horses and cattle with Leptospira interrogans. A total of 600 serum samples from horses and cattle were examined for L. interrogans antibodies using the microscopic agglutination test to determine anti‐Leptospira IgG antibodies against a panel of eight important Leptospira antigens in Iran. Then, serum total antioxidant capacity, catalase, glutathione peroxidase, and malondialdehyde activities, and nitric oxide, total protein, serum amyloid A, haptoglobin, and albumin concentrations were measured in seropositive and seronegative samples. Serum catalase activities and malondialdehyde, serum amyloid A, and haptoglobin ‌concentrations in seropositive cattle and horses were significantly higher (P < .05) than in those that were seronegative. Antibody titers increased from 1:100 to 1:200 in cattle with L. interrogans infection, resulting in a decrease in the serum total antioxidant capacity (P < .05), an increase in serum glutathione peroxidase (P < .01) activity and nitric oxide (P < .05) , serum amyloid A (P < .01), and haptoglobin (P < .05) concentrations. Following the increase in the agglutinating antibody titers in horses infected with L. interrogans, the serum total antioxidant capacity (P < .01) decreased, and serum nitric oxide (P < .05), malondialdehyde (P < .05), and serum amyloid A (P < .01) concentrations were increased. In this study, horses and cattle had extensive changes in oxidant/antioxidant equilibrium and acute‐phase protein concentrations when infected with L. interrogans. We also demonstrated a direct link between antibody titers and the type of leptospiral serovar using serum oxidative and inflammatory markers.