Posttranslational histone modification by acetylation or methylation regulates gene expression. Here, we investigated the role of the histone lysine methyltransferase MLL for angiogenic functions in human umbilical vein endothelial cells. Suppression of MLL expression by siRNA or incubation with the pharmacologic methyltransferase inhibitor 5'-deoxy-5'-(methylthio)adenosine significantly decreased endothelial-cell migration and capillary sprout formation, indicating that methyltransferase activity is required for proangiogenic endothelial-cell functions. Because the expression of homeodomain transcription factors (Hox) is regulated by MLL, we elucidated the role of Hox gene expression. MLL silencing was associated with reduced mRNA and protein expression of HoxA9 and HoxD3, whereas HoxB3, HoxB4, HoxB5, and HoxB9 were not altered. Overexpression of HoxA9 or HoxD3 partially compensated for impaired migration in MLL siRNA-transfected endothelial cells, suggesting that HoxA9 and HoxD3 both contribute to MLL-dependent migration. As a potential underlying mechanism, MLL siRNA down-regulated mRNA and protein levels of the HoxA9-dependent axon guidance factor EphB4. In contrast, MLL knockdown effects on capillary sprouting were not rescued by HoxA9 or HoxD3 overexpression, indicating that MLL affects additional targets required for 3-dimensional sprout formation. We conclude that MLL regulates endothelial-cell migration via HoxA9 and EphB4, whereas sprout formation requires MLL-dependent signals beyond HoxA9 and HoxD3.