N-glycosylation, GPI anchoring, and C-mannosylation are essential post-translational protein modifications that all contain mannose residues transferred from the donor Dolichol-phosphate-mannose (DPM) within the endoplasmic reticulum (ER). DPM is initially generated on the cytosolic side of the ER by transferring mannose from GDP-mannose to the lipid dolichol-phosphate, and then flipped to the ER lumen. Despite more than three decades of research, the mechanism responsible for the luminal translocation of DPM has not been uncovered, although some evidence suggests that a lipid scramblase may be the basis of this process. MPDU1, a member of the PQ-loop protein family resident in the ER, is potentially involved in the translocation reaction of DPM, perhaps being the elusive scramblase, but definitive and convincing evidence supporting this role is lacking. Human MPDU1 loss of function leads to Congenital Disorders of Glycosylation type I f (CDG-If) due to defective biosynthesis of N-glycosyl moieties and GPI anchors and loss of tryptophan C-mannosylation. Understanding the precise role of MPDU1 may open new avenues for developing therapies to control CDG-If. In this study, we identified a putative MPDU1 homologue in T. gondii, named TgMPDU1, which localizes to the ER. Ablation of TgMPDU1 resulted in the loss of C-mannosylation and a decrease in parasite fitness. N-glycosylation was not altered in the TgMPDU1 knock-out strain (ΔMPDU1) because, unlike mammals, N-glycosylation in Toxoplasma does not require mannose in the ER lumen. GPI anchor formation was defective only when ΔTgMPDU1 parasites were grown in conditions at reduce levels of DPM. However, C-mannosylation seemed to be more dependent on MPDU1 activity, as it was defective even when ΔTgMPDU1 parasites were cultured under normal conditions. Overall, these results shed new light on the function of MPDU1, suggesting that this protein might play a role in increasing the efficiency of using DPM as a mannose donor rather than being the scramblase responsible for flipping DPM from the ER cytosol side to the lumen.