We recently reported that apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides inhibit tumor growth and improve survival in a mouse model of ovarian cancer. The current study was designed to examine whether inhibition of angiogenesis is one of the mechanisms for the observed anti-tumorigenic effects. The apoA-I mimetic peptide L-5F had no affect on proliferation and cell viability of human umbilical vascular endothelial cells (HUVECs) in the basal state; however, treatment with L-5F at 1, 3, and 10 μg ml-1, dose-dependently inhibited both vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced proliferation, cell viability, migration, invasion and tube formation in HUVECs. L-5F inhibited VEGF- and bFGF-induced activation of their corresponding receptors, VEGFR2 and FGFR1, as well as downstream signaling pathways, including Akt and ERK1/2. MicroCT scanning and immunohistochemistry staining demonstrated that daily injection of L-5F (10 mg kg-1) decreased both the quantity and size of tumor vessels in mice. L-5F treated mice showed significantly reduced levels of VEGF in both tumor tissue and the circulation, which is consistent with in vitro data showing that L-5F inhibited production and secretion of VEGF from mouse and human ovarian cell lines in the absence and presence of exogenously added lysophosphatidic acid, a potent tumor promoter. In conclusion, our data that L-5F inhibits angiogenesis suggests that apoA-I mimetic peptides may serve as novel anti-angiogenesis agents for the treatment of angiogenesis-associated diseases, including cancer. © The Royal Society of Chemistry.