Oxidative stress (OS) is a major problem during in vitro culture of embryos. Numerous studies have shown that melatonin, which is known to have antioxidant properties, prevents the occurrence of OS in embryos. However, the molecular mechanisms by which melatonin prevents OS in embryos are still unclear. The present study suggests a possible involvement of the nuclear factor erythroid 2-related factor 2/antioxidant-responsive element (Nrf2/ARE) signaling pathway, which is one of the prominent signals for OS prevention through Nrf2 activation, connecting melatonin, OS prevention and porcine embryonic development. The aim of this study was to investigate the effects of melatonin (10-7 M) on porcine embryonic development via the Nrf2/ARE signaling pathway; brusatol (50 nM; Nrf2 specific inhibitor) was used to validate the mechanism. Treatment of porcine embryo with melatonin significantly increased formation rates of blastocysts and their total cell numbers and also upregulated the expression of Nrf2/ARE signaling and apoptosis-related genes (MT2, NRF2, UCHL, HO-1, SOD1 and BCL-2). Furthermore, the expression of proteins (NRF2 and MT2) was also upregulated in the melatonin-treated group. Concomitantly, brusatol significantly inhibited these effects, upregulating the expression of KEAP1 and BAX, including the expression level of KEAP1 protein. These results provide evidences that melatonin prevents OS through Nrf2/ARE signaling pathway in porcine in vitro fertilization -derived embryos.