Oxidative stress with higher levels of leptin and inflammatory response are key processes related to pathogenesis of both T. cruzi infection and aging. Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the expression of several genes implicated in the oxidative stress response in many pathological conditions. Melatonin is a pleiotropic hormone with, antioxidant, anti-inflammatory and anti-aging actions. Then, we hypothesized that Nrf2 response is impaired during the acute T. cruzi (9 days) infection and that melatonin rescues Nrf2 responses. Young (5 weeks-old) and middle-aged (18 months-old) male Wistar rats were infected with T. cruzi. Nrf2 translocation and markers of inflammation and oxidative stress were analyzed in blood and spleen. Increased apoptosis levels and oxidative stress indicators were observed in the rat spleen during T. cruzi infection. These responses were accompanied by decreased Nrf2 expression and increased expression of nuclear factor kappa B (NFκB). Melatonin (5 mg/kg/day; p.o. gavage) attenuated the superoxide anion (O2−) and hydrogen peroxide (H2O2) production induced by T. cruzi infection. Increased expressions of catalase and superoxide dismutase (SOD) were detected in the spleen of melatonin-treated rats infected with T. cruzi. Melatonin treatment inhibited the spleen NF-κB activation and downregulates the levels of circulating interleukin (IL)-4, IL-10 and tumor necrosis factor (TNF)-α in T. cruzi middle-aged infected rats. Increased levels of the chemokine CXCL1 in middle-aged control rats was observed, confirming that aging alters the production of this chemokine. In T. cruzi infected young animals, CXCL1 was up-regulated when compared to non-infected young ones. For young or middle-aged animals, melatonin treatment had no significant effect on CXCL1 levels. Our findings demonstrate an important role for Nrf2/NF-kB regulation as a possible mechanism by which melatonin attenuates oxidative stress, and provide new insights for further studies of this indoleamine as a therapeutic co-adjuvant agent against T. cruzi infection. Display omitted •Melatonin regulates Nrf2/NF-kB response during acute T. cruzi infection;•The induction of Nrf2 regulation by melatonin was linked to improved Nrf2 nuclear translocation;•Treatment with melatonin reduced NF-κB expression in middle-aged treated animals, infected or not;•Melatonin regulates CD4+T and CD8+ T lymphocytes and splenocyte apoptosis.