AbstractMultidrug-resistant bacterial pathogens are a major medical concern. E. coli, particularly the pathotype extraintestinal pathogenic E. coli (ExPEC), is a leading cause of bloodstream infections. As natural parasites of bacteria, bacteriophages are considered a possible solution to treat patients infected with antibiotic resistant strains of bacteria. However, the development of phage as an anti-infective therapeutic is hampered by limited knowledge of the physiologic factors that influence their properties in complex mammalian environments such as blood. To address this barrier, we tested the ability of phage to kill ExPEC in human blood. Phages are effective at killing ExPEC in conventional media but are substantially restricted in this ability in blood. This phage killing effect is dependent on the levels of free metals and is inhibited by the anticoagulant EDTA. The EDTA-dependent inhibition of ExPEC killing is overcome by exogenous iron, magnesium, and calcium. Metal-enhanced killing of ExPEC by phage was observed for several strains of ExPEC, suggesting a common mechanism. The addition of metals to a murine host infected with ExPEC stimulated a phage-dependent reduction in ExPEC levels. This work defines a role for circulating metals as a major factor that is essential for the phage-based killing of bacteria in blood.