Grape-derived polyphenols, particularly those found in Concord grape juice (CGJ), are known for their vascular protective effects, partly attributed to their ability to directly influence blood vessels by enhancing the production of nitric oxide (NO) by endothelial cells. This study aimed to investigate whether CGJ, rich in polyphenols, induces endothelial nitric oxide synthase (eNOS) expression in porcine coronary artery endothelial cells and to elucidate the underlying signaling mechanisms. CGJ treatment led to dose- and time-dependent increases in both eNOS mRNA and protein levels, accompanied by elevated NO production in endothelial cells. The upregulation of eNOS mRNA by CGJ was not associated with enhanced mRNA stability but was inhibited by antioxidants such as MnTMPyP, PEG-catalase, and catalase. Additionally, inhibitors of PI3-kinase (wortmannin), p38 MAPK (SB 203580), and JNK (SP 600125) attenuated the stimulatory effect of CGJ on eNOS expression. CGJ also induced the generation of reactive oxygen species (ROS) in endothelial cells, which was mitigated by antioxidants. Furthermore, CGJ triggered the phosphorylation of p38 MAPK and JNK kinases, processes blocked by MnTMPyP. Phosphorylation of the transcription factors FoxO1 and FoxO3a, known negative regulators of eNOS expression, was induced by CGJ and inhibited by MnTMPyP, PEG-catalase, wortmannin, SB 203580, and SP 600125. Chromatin immunoprecipitation assays revealed that CGJ caused dissociation of FoxO3a from the eNOS promoter, suggesting a mechanism by which CGJ prevents FoxO-mediated repression of eNOS gene expression. In conclusion, CGJ enhances eNOS expression and NO production in endothelial cells through a redox-sensitive mechanism involving PI3-kinase/Akt, p38 MAPK, and JNK signaling pathways, and by inhibiting SP 600125 negative control FoxO1 and FoxO3a transcription factors.