Background: Hospital-acquired anemia (HAA) during acute myocardial infarction (AMI) is associated with higher mortality and worse health status and often develops in the absence of recognized bleeding. The extent to which diagnostic phlebotomy, a modifiable process of care, contributes to HAA is unknown. Methods: We studied 17 676 patients with AMI from 57 US hospitals included in a contemporary AMI database from January 1, 2000, through December 31, 2008, who were not anemic at admission but developed moderate to severe HAA (in which the hemoglobin level declined from normal to <11 g/dL), a degree of HAA that has been shown to be prognostically important. Patients'total diagnostic blood loss was calculated by multiplying the number and types of blood tubes drawn by the standard volume for each tube type. Hierarchical modified Poisson regression was used to test the association between phlebotomy and moderate to severe HAA, after adjusting for site and potential confounders. Results: Moderate to severe HAA developed in 3551 patients( 20%). The mean (SD) phlebotomy volume was higher in patients with HAA (173.8 [139.3]mL) vs those without HAA (83.5 [52.0mL];P<.001). There was significant variation in the mean diagnostic blood loss across hospitals (moderate to severe HAA: range, 119.1-246.0 mL; mildHAAor no HAA: 53.0-110.1 mL). For every 50mL of blood drawn, the risk of moderate to severe HAA increased by 18% (relative risk [RR], 1.18;95% confidence interval [CI],1.13-1.22), which was only modestly attenuated after multivariable adjustment (RR, 1.15; 95% CI, 1.12-1.18). Conclusions: Blood loss from greater use of phlebotomy is independently associated with the development of HAA. These findings suggest that HAA may be preventable by implementing strategies to limit blood loss from laboratory testing.