Abstract: Sphingosine kinase 1 (SPHK1) is a newly discovered modulator of hypoxia inducible factor 1α (HIF-1α) with various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, the biological mechanisms of melatonin were elucidated in association with SPHK1 pathway in PC-3 prostate cancer cells under hypoxia. Melatonin inhibited the stability of HIF-1α in a time- and concentration- dependent manners. Also, melatonin decreased SPHK1 activity in PC-3 cells during hypoxia. Furthermore, melatonin suppressed AKT/glycogen synthase kinase-3β (GSK-3β) signaling pathway, which stabilizes HIF-1α via inhibition of von Hippel-Lindau tumor suppressor protein. Consistently, siRNA-SPHK1 and sphingosine kinase inhibitor (SKI) effectively blocked the expression of HIF-1α, phospho-AKT and vascular endothelial growth factor (VEGF) production in PC-3 cells under hypoxia, suggesting the role of SPHK1 in melatonin-inhibited HIF-1α accumulation. Moreover, reactive oxygen species (ROS) scavenger N-acteylcysteine enhanced melatonin-inhibited HIF-1α expression and SPHK1 activity. Overall, our findings suggest that melatonin suppresses HIF-1α accumulation via inhibition of SPHK1 pathway and ROS generation in PC-3 cells under hypoxia.