Trypanosoma vivax infection is an emerging condition that causes damage and mortality among cattle and is transmitted by mechanical vectors or contaminated fomites. This disease has been spreading in southern Brazil, causing anemia, weight loss, diarrhea, abortion, and infertility; however, its behavior and host–parasite relationships are not yet fully understood. To clarify this issue, animals that presented clinical signs were subjected to an immunochromatographic screening test. An indirect immunofluorescence test was then performed on samples collected before treatment (the gold standard test), which showed that in the herd of 20 cows, we had 14 seropositive for T. vivax. Blood samples were collected before and after treatment to study the effects of the disease and treatment, with the cows divided into two groups: infected and uninfected. Cows were evaluated for hematologic, biochemical, and antioxidant responses, comparing them with uninfected and infected animals, as well as pre- and post-treatment (isometamidium chloride—1 mg/kg body weight [BW]). There was no difference (p > 0.05) between groups in milk production and feed intake; however, ten days after treatment, there was an increase of 1.72 kg of milk in cows diagnosed as infected with T. vivax. Seropositive cows had lower erythrocyte counts, hemoglobin concentrations, hematocrit, platelet counts, and lymphocyte and granulocyte counts. In seropositive cows, the higher total protein concentration is due to increased globulins, which the protein profile by electrophoresis showed to be related to higher levels of immunoglobulins (IgA and other heavy-chain immunoglobulins), ceruloplasmin, haptoglobin, ferritin, C-reactive protein; associated with lower transferrin levels. The activity of the enzymes aspartate aminotransferase, gamma-glutamyl transferase, cholinesterases, and creatine kinase were compared in seronegative and seropositive cows for T. vivax. Lower serum calcium levels were observed in seropositive cows. Cows diagnosed with trypanosomosis presented high levels of reactive oxygen species, lipid peroxidation, protein oxidation, nitrite/nitrate activity, superoxide dismutase, and glutathione peroxidase. The enzymes catalase and glutathione S-transferase presented lower activity in the blood of seropositive cows compared to the control on the day of diagnosis, which was no longer observed ten days after treatment. The animals exhibited hypogalactia, anemia, thrombocytopenia, leukopenia, and acute phase response accompanied by liver and muscle tissue damage and oxidative stress, demonstrating the effect of T. vivax infection in naturally infected Jersey cows.