Background: Salidroside is the major active component of Rhodiola rosea, a traditional Chinese herbal medicine used for protection against ultraviolet (UV) radiation. Objectives: This study investigated whether salidroside can protect skin from ultraviolet B (UVB)-induced oxidative damage in human immortalized HaCaT keratinocytes and the skin of guinea pigs. Methods: Using HaCaT cell models, the effects of salidroside on oxidative damage and possible regulatory factors [including NF-E2-related factor 2 (Nrf2), NAD(P) H-quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1)] were examined. In addition, the regulatory effects of salidroside on apoptotic sunburn cells (SBCs) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive epidermal cells on UVB-exposed guinea pig skin were also investigated. Results: We found that salidroside pretreatment upregulated Nrf2 translocation to the nucleus and transcription activity in HaCaT cells, as reflected by the increased nuclear accumulation of Nrf2 as well as the gene and protein expression of downstream Nrf2 antioxidants, including NQO1 and HO-1. In addition, we also found that pretreatment with salidroside reactive oxygen species (ROS) in irradiated HaCaT cells. The oral administration of salidroside (0.1% w/w) to guinea pigs inhibited the UVB-mediated formation of apoptotic SBCs and 8-OHdG-positive epidermal cells in the skin of guinea pigs. Conclusions: Our results show that UVB-induced oxidative damage can be prevented by salidroside with upregulation of nuclear Nrf2 expression.