Galectins are β-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (Nφ) from C57BL/6 wild-type (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MTT assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in Nφ culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in Nφ cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Nφ number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates T. gondii-induced Nφ toxicity as well as Nφ degranulation regardless of infection. Furthermore, Gal-3 expression by Nφ was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-α later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in Nφ following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in Nφ life span and activation during early T. gondii infection.