We have recently reported that keratin 14‐promoter‐driven vascular endothelial growth factor (VEGF)‐E_NZ‐7 transgenic mice have a significant number of capillary vessels in subcutaneous tissue. However, these vessels are generated in a layer some distance from the epithelial basal cells that express VEGF‐E_NZ‐7, suggesting that one or more antiangiogenenic molecules may exist very near the basal cell layer. By screening keratinocyte‐conditioned medium, we found that thrombospondin‐1 (TSP‐1) is produced from keratinocytes and suppresses human umbilical vein endothelial cells (HUVEC) growth as well as tubular formation in a HUVEC–fibroblast coculture system. Different to the known mechanism of CD36‐dependent endothelial cell apoptosis, the HUVEC we used did not express CD36 at detectable levels, indicating a new mechanism for TSP‐1‐induced antiangiogenesis. We found that TSP‐1 induces little apoptosis of endothelial cells but causes cell‐cycle arrest, increasing the amounts of p21^CIP/WAF‐1 and unphosphorylated retinoblastoma (Rb) in HUVEC. CD36‐binding peptide in TSP‐1 and CD36‐neutralizing antibody did not block the TSP‐1‐induced cell‐cycle arrest. Our results strongly suggest that TSP‐1 utilizes a novel pathway for its antiangiogenic effect independent of CD36, and suppresses the cell cycle. (Cancer Sci 2007; 98: 1491–1497)