Tumour therapy with cyclophosphamide (CPA), an alkylating chemotherapeutic agent, has been associated with reduced tumour blood supply and antiangiogenic effects when applied in a continuous, low-dose metronomic schedule. Compared to conventional high-dose scheduling, metronomic CPA therapy exhibits antitumoural activity with reduced side effects. We have studied potential antiangiogenic properties of acrolein which is released from CPA after hydroxylation. Acrolein adducts were found in tumour cells and tumour endothelial cells of CPA-treated mice, suggesting an in vivo relevance of acrolein. In vitro, acrolein inhibited endothelial cell proliferation, endothelial cell migration and tube formation. Moreover, acrolein caused disassembly of the F-actin cytoskeleton and inhibition of αvβ3 integrin clustering at focal adhesions points in endothelial cells. Acrolein treatment modulated expression of thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis known to be linked to antiangiogenic effects of metronomic CPA therapy. Further on, acrolein treatment of primary endothelial cells modified NF-κB activity levels. This is the first study that points at an antiangiogenic activity of acrolein in metronomically scheduled CPA therapy.