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Due to the limited effectiveness of existing drugs for the treatment of toxoplasmosis, there is a dire need for the discovery of new therapeutic options. Artemether is an important drug for malaria and several studies have indicated that it also exhibits anti-<i>T. gondii</i> activity. However, its specific effect and mechanisms are still not clear. To elucidate its specific role and potential mechanism, we first evaluated its cytotoxicity and anti-<i>Toxoplasma</i> effect on human foreskin fibroblast cells, and then analyzed its inhibitory activity during <i>T. gondii</i> invasion and intracellular proliferation. Finally, we examined its effect on mitochondrial membrane potential and reactive oxygen species (ROS) in <i>T. gondii</i>. The CC₅₀ value of artemether was found to be 866.4 μM, and IC₅₀ was 9.035 μM. It exhibited anti-<i>T. gondii</i> activity and inhibited the growth of <i>T. gondii</i> in a dose-dependent manner. We also found that the inhibition occurred primarily in intracellular proliferation, achieved by reducing the mitochondrial membrane integrity of <i>T. gondii</i> and stimulating ROS production. These findings suggest that the mechanism of artemether against <i>T. gondii</i> is related to a change in the mitochondrial membrane and the increase in ROS production, which may provide a theoretical basis for optimizing artemether derivatives and further improving their anti-<i>Toxoplasma</i> efficacy.