Wine intake affects the antioxidant enzyme activities that contribute to the overall antioxidant properties of wine. The purpose of this study is to evaluate whether alcohol-free wine has any effect on antioxidant enzymes. The study was a randomized cross-over human intervention. A low phenolic diet (LPD) was designed to prevent interference from polyphenols in other food sources. In the first period, the volunteers ate only this low phenolic diet; in the second, they ate this diet and also drank 300 mL of alcohol-free wine. The enzymes under study were: superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The activities of glutathione reductase, superoxide dismutase and catalase decreased during the LPD period and increased in the LPD+dealcoholized wine period. On the third day of intervention, significant changes were observed in glutathione reductase and superoxide dismutase activity for both intervention periods under study. Catalase activity changed significantly on the seventh day of intervention. Antioxidant enzymes modulated their activity more easily than the endogenous antioxidants, which did not undergo any changes. Our results show for the first time that the increase in the activity of the antioxidant enzymes is not due to the alcohol content in wine but to the polyphenolic composition. Therefore, alcohol-free wine could be an excellent source of antioxidants to protect people suffering from oxidative stress (cancer, diabetes, alzheimer, etc.) who should not consume alcohol.