Angiogenesis plays a vital role in tumor progression and metastasis. To better understand the role of anesthesia in tumor biology, we previously reported that bupivacaine displayed the inhibitory effects in endothelial cells. In this work, we demonstrated that fentanyl, an opioid medication commonly used in cancer patients, stimulated tumor angiogenesis. We found that fentanyl at nanomolar concentrations significantly stimulated capillary network formation of human lung tumor-associated endothelial cell (HLT-EC) in a similar manner as vascular endothelial growth factor (VEGF), and furthermore that the stimulatory effect of fentanyl was mainly involved in early stage of HLT-EC vascular structure assembly. Particularly, fentanyl significantly increased HLT-EC growth and migration. Fentanyl also protected HLT-EC from apoptosis induced by growth factor withdrawal. In contrast, the same concentrations of fentanyl did not affect human lung cancer cell growth and survival. Fentanyl stimulated migration of some but not all tested human lung cancer cells. Mechanism analysis suggested that fentanyl activates multiple proangiogenic signaling pathways, including VEGFR2/FAK/PI3K/Akt and small GTPases. Our work systematically demonstrates that fentanyl stimulates tumor angiogenesis via activating multiple pro-angiogenic signaling pathways. Our findings highlight the potential adverse effect of fentanyl in cancer patients. (C) 2020 Elsevier Inc. All rights reserved.