A grape pomace extract enhanced antioxidant mechanisms in muscle and endothelial cells both in the absence and in the presence of oxidative stress-induced agent tert-butyl hydroperoxide (tBHP). In particular, muscle (C2C12) and endothelial (EA.hy926) cells were treated with the extract at noncytotoxic concentrations for 24 h, and the oxidative stress markers, total reactive oxygen species (ROS), glutathione (GSH), thiobarbituric reactive substances (TBARS), and protein carbonyl levels were assessed. The results showed that the grape extract treatment reduced significantly ROS, TBARS, and protein carbonyl levels and increased GSH in C2C12 cells, while it increased GSH and decreased protein carbonyl levels in EA.hy926 cells. In the presence of tBHP, the grape extract treatment in C2C12 cells reduced significantly ROS, TBARS, and protein carbonyls and increased GSH compared with tBHP alone treatment, while, in EA.hy926 cells, the extract decreased significantly TBARS and protein carbonyls but increased GSH. The antioxidant potency of the extract was different between muscle and endothelial cells suggesting that the antioxidant activity depends on cell type. Moreover, the antioxidant activity of the grape extract, in both cell lines, exerted, at least in part, through increase in GSH levels. The present work is the first to report the effects of grape extract shown for skeletal muscle cells.