Ginsenosides are major bioactive constituents that are responsible for the diverse pharmacological activities of ginseng. This work aimed to assess the skin anti-photoaging activities of the two stereoisomeric forms of ginsenoside Rg3, 20( S )-Rg3 and 20( R )-Rg3. When the two Rg3 stereoisomers were added to cultured human keratinocyte HaCaT cells prior to irradiation with 70 mJ/cm² UV-B, 20( S )-Rg3, but not 20( R )-Rg3, decreased the UV-B-induced intracellular reactive oxygen species (ROS) levels in a concentration-dependent manner, as detected by both fluorometric and confocal microscopic analyses. Likewise, 20( S )-Rg3, but not 20( R )-Rg3, decreased the UV-B-induced ROS levels in human dermal fibroblast cells. Both stereoisomers were unable to modulate the nitric oxide levels in HaCaT cells under UV-B irradiation, and induced no cytotoxicity in cultured keratinocytes and fibroblasts. 20( S )-Rg3 suppressed the UV-B-induced matrix metalloproteinase (MMP)-2 activities in HaCaT cells. Taken together, these results indicate that 20( S )-Rg3 possesses both ROS-scavenging and MMP-2 inhibitory activities, while 20( R )-Rg3 possesses neither activity. These findings imply that ginsenoside Rg3 stereoselectively demonstrates skin anti-photoaging activities.