Introduction: Toxoplasmosis is an alarming public health problem that affects more than one-third of the global population. We investigated the antiparasitic effects of MjTX-II, a Lys-49-PLA₂ from Bothrops moojeni, in human trophoblast cells and villous explants. Methods: MjTX-II was purified using two ion-exchange chromatographic steps and characterized through biochemical and proteomic analyses. The BeWo cells' viability was assessed using an MTT assay. The impact of MjTX-II on T. gondii was evaluated via trypan blue staining and scanning electron microscopy. The effect on the parasite's adhesion and invasion was assessed via differential antibody staining. The antiproliferative effect was evaluated using a β-galactosidase assay. Cytokine and VEGF levels were measured using ELISA and a cytometric bead array. ROS concentrations were quantified according to the intracellular peroxide-dependent oxidation. The involvement of VEGFR2 and ICAM-1 in the antiparasitic mechanism was investigated through docking simulations. Results: MjTX-II exhibited a high degree of purity in the biochemical and proteomic analyses. The MTT assay showed that MjTX-II did not reduce the viability of the BeWo cells. MjTX-II affected the surface of the parasites, decreased the number of intracellular parasites, and inhibited their intracellular proliferation. MjTX-II modulated the cellular environment by increasing the levels of IL-6, MIF, and ROS and reducing the levels of VEGF. The anti-T. gondii activity involved the modulation of the ROS and VEGF levels, as the addition of the ROS scavenger NAC or rVEGF reduced the antiprotozoal effect. Bioinformatic analyses revealed that MjTX-II is able to interact with the extracellular domain of VEGFR2 in the VEGF binding site, as well as ICAM-1, which was corroborated by the immunofluorescence analysis. Conclusions: Non-toxic concentrations of MjTX-II impaired T. gondii infection by increasing the ROS and VEGF levels in the host cells and may have involved interactions with ICAM-1 and VEGFR2, highlighting MjTX-II as prominent tool for the development of effective molecules against toxoplasmosis.