Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract leading to trichomoniasis, the most common sexually-transmitted non-viral disease worldwide. The immune response plays a critical role in the host defense against this parasite. Trichomonas' DNA contains unmethylated CpG motifs (CpGDNA) that in other microorganisms act as modulators of the immune response. However, the molecular mechanisms responsible for CpGDNA immune modulation are still unclear. As macrophages participate in the first line of defense against infection, we investigated the type of immune response of murine macrophages to T. vaginalis DNA (TvDNA). We observed high expression of the proinflammatory cytokines IL-6 and IL-12p40 in macrophages stimulated with TvDNA. In contrast, the anti-inflammatory response, assessed by IL-10 and IL-13 mRNA expression was delayed. This suggests that the immune response induced by TvDNA is modulated through cytokine production, mediated partly by NADPH-oxidase activity, as TvDNA induced reactive species of oxygen production and a rounded morphology in macrophages indicative of an M1 phenotype. Furthermore, infected mice pretreated with TvDNA displayed persistent vulvar inflammation and decreased parasite viability consistent with higher proinflammatory cytokine levels during infection compared to untreated mice. Overall, our findings suggest that TvDNA pretreatment modulates the immune response favouring parasite elimination.