We have previously reported that MAP Kinase Phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in human umbilical vein endothelial cells (EC). We now show that vascular endothelial growth factor (VEGF) is another efficacious activator of MKP-1 expression in EC. VEGF-A and VEGF-E maximally induced MKP-1 expression in EC; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies we determined that VEGF induced MKP-1 specifically through VEGF Receptor 2 (VEGFR-2) leading to the downstream activation of c-Jun N-terminal protein kinase (JNK). The VEGF-A₁₆₅ isoform stimulated MKP-1 expression; whereas, VEGF-A₁₆₂ induced the gene to a lesser extent and VEGF-A₁₂₁ isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 co-receptors, blocked MKP-1 induction. The Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression; whereas, Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK; whereas, extracellular-signal-regulated protein kinase (ERK) was critical for thrombin-induced MKP-1 expression. In EC treated with siRNA targeting MKP-1, JNK, ERK and p38 phosphorylation was prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1 null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a trans-well assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in EC and point to the importance of MKP-1 induction in VEGF-stimulated EC migration.