Infarction occurs when myocardial perfusion is interrupted for prolonged periods of time. Short episodes of ischemia and reperfusion protect against tissue injury when the heart is subjected to a subsequent prolonged ischemic episode, a phenomenon known as ischemic preconditioning (IPC). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to hypoxia/ischemia and is required for IPC. In this study, we performed a cellular and molecular characterization of the role of HIF-1 in IPC. We analyzed mice with knockout of HIF-1α or HIF-1β in Tie2(+) lineage cells, which include bone marrow (BM) and vascular endothelial cells, compared with control littermates. Hearts were subjected to 30 min of ischemia and 120 min of reperfusion, either as ex vivo Langendorff preparations or by in situ occlusion of the left anterior descending artery. The IPC stimulus consisted of two cycles of 5-min ischemia and 5-min reperfusion. Mice lacking HIF-1α or HIF-1β in Tie2(+) lineage cells showed complete absence of protection induced by IPC, whereas significant protection was induced by adenosine infusion. Treatment of mice with a HIF-1 inhibitor (digoxin or acriflavine) 4 h before Langendorff perfusion resulted in loss of IPC, as did administration of acriflavine directly into the perfusate immediately before IPC. We conclude that HIF-1 activity in endothelial cells is required for acute IPC. Expression and dimerization of the HIF-1α and HIF-1β subunits is required, suggesting that the heterodimer is functioning as a transcriptional activator, despite the acute nature of the response.