Objective To explore the role of CD73 in Toxoplasma gondii (T. Gondii) infection; To verify the host cells absent with CD73 would have less infection and replication of T. Gondii. Methods CD73 knockout and wild-type mice were infected with 20 cysts of ME 49 by oral gavage. The survival rate, clinic symptoms, pathology in the gut, and cytokine production were investigated in infected mice. Macrophages and dendritic cells with CD73-/- and CD73+/+ were infected with RH-YFP. The infective rates, the number of intracellular RH-YFP in the host cells, and the replication of intracellular RH-YFP were detected in these two type cells. Results CD73 knockout mice had a higher survival rate and milder clinic presentation with less morphologies in the gut in the acute T. Gondii infection compared with wild-type mice. CD73-/- macrophages and dentritic cells had less T. Gondii infection, and less intracellular parasite number and replication compared with CD73+/+ macrophages and dentritic cells in vitro. Conclusions CD73 as a GPI-anchored surface protein of host cell might be involved in forming the parasite vacuole, and promote the parasite's attachment and invasion of host eeUs. Host cells absence with CD73 would be less infection and replication of T. Gondii, and relative resistant parasites infection.