The infectivity and virulence of pathogenic trypanosomatids are directly associated with the efficacy of their antioxidant system. Among the molecules involved in the trypanosomatid response to reactive oxygen or nitrogen species, trypanothione reductase (TRed) is a key enzyme. In this study, we performed a molecular and functional characterization of the TRed enzyme from Trypanosoma rangeli (TrTRed), an avirulent trypanosome of mammals. The TrTRed gene has an open reading frame (ORF) of 1473 bp (~490 aa, 53 kDa) and occurs as a single-copy gene in the haploid genome. The predicted protein contains two oxidoreductase domains, which are equally expressed in the cytosol of epimastigotes and trypomastigotes. Nicotinamide adenine dinucleotide phosphate (NADPH) generation is reduced and endogenous H2O2 production is elevated in T. rangeli Choachí strain compared with T. cruzi Y strain epimastigotes. Oxidative stress induced by H2O2 does not induce significant alterations in TrTRed expression. Overexpression of TrTRed did not influence in vitro growth or differentiation into trypomastigotes, but mutant parasites showed increased resistance to H2O2-induced stress. Our results indicate that T. rangeli constitutively expresses TRed during the entire life cycle, with reduced levels during infective and non-replicative trypomastigote stages.