Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as clopidogrel (Plavix); however, very little is known about the ADP-preferring nucleotide receptors (P2Y₁, P2Y₁₂, and P2Y₁₃) in endothelium. Here, we show that ADP stimulates migration of cultured human umbilical vein endothelial cells (HUVECs) in both Boyden chamber and in vitro wound repair assays. This promigratory effect was mimicked by 2-MeSADP, but not by AMP, and was inhibited by MRS2179 (P2Y₁ receptor antagonist) but not by AR-C69931MX (P2Y_12/13 receptor antagonist). RT-PCR revealed abundant P2Y₁, barely detectable P2Y₁₂, and absent P2Y₁₃ receptor message in these cells. In addition, both ADP and 2-MeSADP, but not AMP, activated the mitogen-activated protein kinase pathways as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 kinase. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of such transcription factors downstream of the JNK and p38 pathways as c-Jun and activating transcription factor-2. These signaling events were inhibited by MRS2179 but not by AR-C69931MX. Furthermore, blockade of the ERK or JNK pathways by U0126 and SP600125, respectively, abolished ADP- and 2-MeSADP–stimulated HUVEC migration. However, inhibition of the p38 pathway by SB203580 partially suppressed ADP- and 2-MeSADP–induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y₁ receptor–mediated MAPK pathways, possibly contributing to reendothelialization and angiogenesis after vascular injury.