ABSTRACTMounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the rich media used forin vitroculture contains supraphysiological nutrient concentrations, pathogens encounter a relatively restrictive environmentin vivo. We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically relevant mild nutrient stress (or “metabolic priming”) triggers increased tolerance of a potent antimalarial drug. We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in metabolism or using a media formulation that simulates the human plasma environment. We determined that priming-induced tolerance was restricted to parasites that had newly invaded the host red blood cell, but the effect was not dependent on genetic background. The molecular mechanisms of this intrinsic effect mimic aspects of genetic tolerance, including translational repression and protein export. This finding suggests that regardless of the impact on survival rates, environmental stress could stimulate changes that ultimately directly contribute to drug tolerance. Because metabolic stress is likely to occur more frequentlyin vivocompared to the stablein vitroenvironment, priming-induced drug tolerance has ramifications for howin vitroresults translate toin vivostudies. Improving our understanding of how pathogens adjust their metabolism to impact survival of current and future drugs is an important avenue of research to slow the evolution of resistance.