Angiogenesis, the formation of new capillaries from preexisting vessels, plays an essential role in revascularization of the myocardium following myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart following MI, is shown to be essential for angiogenesis in the invasiveness of tumor cells, the progression of arthritic conditions and endometriosis, and the promotion of wound healing. Here we studied the steps of angiogenesis in response to IL-1β in cardiac microvascular endothelial cells (CMECs) and aortic tissue. Cell cycle progression analysis using flow cytometry indicated a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells. IL-1β significantly reduced levels of fibrillar actin in the cytoskeleton, a pre-requisite for tube formation, as indicated by phalloidin-FITC staining. Wound healing assays demonstrated IL-1β prevents cell-to-cell contact formation. On the other hand, vascular endothelial growth factor-D (VEGF-D) initiated restoration of the cell monolayer. IL-1β significantly inhibited in vitro tube formation as analyzed by three-dimensional collagen matrix assay. Aortic ring assay demonstrated that IL-1β inhibits basal and VEGF-D-stimulated microvessel sprouting from aortic rings. The data presented here are novel and of significant interest, providing evidence that IL-1β impedes the process of angiogenesis in myocardial endothelial cells.