Melatonin can be synthesized and secreted not only by the pineal gland but also by many extra-pineal tissues. It has been shown that many ovarian functions are regulated by melatonin locally. Ovarian hyperstimulation syndrome (OHSS) is a serious complication during ovulation induction of the in vitro fertilization treatment. To date, the etiology of OHSS is not fully understood. However, vascular endothelial growth factor (VEGF) is recognized as a critical mediator for the pathogenesis of OHSS. High expression of melatonin has been detected in the follicular fluid of OHSS patients. However, whether VEGF expression can be regulated by melatonin in human granulosa cells and further contributes to the pathogenesis of OHSS remain unknown. In this study, we show that melatonin stimulates VEGF expression in human granulosa-lutein (hGL) cells. Our results reveal that the MT2 receptor and activation of AKT are involved in melatonin-induced VEGF expression. Using a rat OHSS model, we report that the VEGF levels are up-regulated in the ovaries of OHSS rats. Blocking the melatonin system by administrating MT2 receptor antagonist, 4-P-PDOT, alleviates OHSS symptoms by decreasing the expression of VEGF. In addition, the expression levels of melatonin and VEGF in the follicular fluid of OHSS patients are up-regulated and positively correlated. This study demonstrates an important role for melatonin in regulating the pathogenesis of OHSS.