Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects on P. falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S. cerevisiae growth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. An hnm1Delta yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound. Based on these data for S. cerevisiae, labeling studies in P. falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies in P. falciparum and in vitro studies with recombinant P. falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity.