Partially purified paeoniflorin (PF), a new cosmetic ingredient from roots of Paeoniae lactiflora, has been developed. Its paeoniflorin content is about 64%, far higher than that of conventional, cosmetic-grade peony root extracts (approximately 10%). In this report, we studied the effects of PF on UV-induced DNA damage in both cultured human keratinocytes and hairless mouse skin. We also investigated the anti-wrinkle effects of PF-containing cosmetic preparations on human skin. From the in vitro and in vivo comet assay, it was revealed that PF protected cells from DNA damage induced by ultraviolet B (UVB) irradiation in both cultured normal human keratinocytes (19.4% decrease at 0.001%) and hairless mouse skin keratinocytes (41% decrease at 0.01%). An eight-week clinical trial using 0.5% PF-containing formulation with 20 volunteers resulted in a statistically significant reduction in facial wrinkles (p < 0.05). These results suggest that the partially purified paeoniflorin has potent anti-aging and anti-wrinkle activities and should be a useful ingredient for these purposes.