In order to discover ingredients for wrinkle-care cosmetics, we prepared 70% ethanol extract from Gynostemma pentaphyllum and examined its activity on collagen synthesis using fibroblast HDFn cells. The G. pentaphyllum extract induced the production of type I procollagen in a dose-dependent manner without showing cell toxicity. The active constituents were isolated from the extract by solvent fractionation and chromatographic purification procedures. NMR data and literature studies led to determine the two isolated compounds as the flavonoid glycosides such as ombuine 3-O-rutinoside (1) and quercetin 3-O-rutinoside (2). The activity screening tests showed that the isolates 1 and 2 induced the production of type I procollagen in a dose-dependent manner. These results suggested that G. pentaphyllum extract containing the flavonoids 1 and 2 could be useful as an active ingredient for wrinkle-care cosmetics.