Homeobox (HOX) family genes, major transcription factors for embryonic development, have been also implicated in vascular development and angiogenesis, particularly with regulation of genes involved in cell–cell or cell–extracellular matrix (ECM) interactions. However, the cellular and molecular functions of HOXD1 in endothelial cells (ECs) are yet to be explored. We here report that HOXD1 is prominently expressed in human ECs and regulates angiogenic activities. Knockdown of HOXD1 in ECs resulted in significant inhibition of migration and adhesion as well as tube like structure formation. These effects were correlated with the reduced expression of integrin β1 (ITGB1), an important signaling component of angiogenesis. Consistently, ITGB1 promoter activity was decreased by HOXD1 knockdown in ECs. Furthermore, we identified the putative HOXD1-binding sites in the promoter region of ITGB1. Together, these findings suggest that HOXD1 plays a significant role in EC functions by regulating the expression of ITGB1.