Oxidative stress is an important cause of skin injury induced by UVB radiation. Moringa oleifera also known as horseradish tree or drumstick tree, have multiple nutraceutical or pharmacological functions. However, whether Moringa oleifera protects skin against oxidative stress injury remains unknown. To investigate the effects of the ethanol extract of Moringa oleifera stem (MSE) on skin oxidative stress injury and its molecular mechanism, we first determined the effect of MSE on epidermal oxidative stress injury induced by H₂O₂ in keratinocytes (HaCaT cells) and by UVB-radiation in mice. Then we investigated the effect of MSE on the enhancement of antioxidant system and activation of PPARα in vitro and in vivo. Furthermore, the flavonoids compositions in MSE were assayed by high-performance liquid chromatography (HPLC), and then molecular docking study was used to assess the major component in MSE to activate PPARα. Our results indicate that MSE (100–400 μg/mL) protected the epidemic cell against oxidative stress injury in vitro and topical treatment with MSE cream (6%) inhibit UVB-induced oxidative stress injury in the epidermis of the mouse skin. PPARα activation is involved in the protective effect of MSE. HPLC assay and molecular docking study indicated that rutin might be the main component in MSE to activate PPARα. These results confirm that MSE exerts the protective effect on oxidative stress induced skin keratinocytes injury. Moreover, the protective effect of MSE is mediated by enhancement of antioxidant defense systems and activation of PPARα in skin keratinocytes.