Roundup 3 plus(®), a glyphosate-based herbicide, is widely used in the ground, but its extensive use has posed a health risk in man. The aim of this study was firstly to investigate how glyphosate alone or included in Roundup 3 plus(®) affected the antioxidant defense system and lipid peroxidation of human cutaneous cells, and secondly, to evaluate the ameliorating effects of antioxidants, as Vitamin C (VitC) and Vitamin E (VitE), against Roundup 3 plus(®)-induced epidermal antioxidant impairment. Our results showed that glyphosate alone or included in Roundup 3 plus(®), induced significant changes in cellular antioxidant status as a glutathione depletion, enzymatic (catalase, glutathione-peroxidase and superoxide dismutase) disorders, and increased lipid peroxidation. VitC or VitE supplementation increased superoxide dismutase, glutathione-reductase and -peroxidase activities and reduced lipid peroxidation in Roundup 3 plus(®)-treated keratinocytes. These in vitro data indicated that VitC and VitE might have preventive effects against deleterious cutaneous cell damage caused by Roundup 3 plus(®).