ABSTRACTIn humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the familiesTrichostrongylidae,Dictyocaulidae,Chabertiidae,Ancylostomatoidea, andAscarididae. We characterized these putative PMTs and found that they possessbona fidePMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using anin vitrophosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested againstHaemonchus contortususing larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates ofH. contortus, with IC50values (95% confidence interval) of 4.30 μM (2.15–8.28), 4.46 μM (3.22–6.16), 28.7 μM (17.3–49.5), and 0.65 μM (0.21–1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potentin vitroanthelmintic activity.