Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis , the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli , based on a disulfide-sensitive β-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.