In order to investigate the mechanism of glycolic acid (GA) function in human stratum corneum, we monitored changes in cathepsin D-like (CD) and chymotrypsin-like (SCCE) proteinases for 3 weeks following topical GA application (50% w/v, pH 0.9) for 30 min to human skin. In the early phase, weakened stratum corneum cohesion in the lower layers was observed on day 2 and the amount of active CD in the upper layer of the stratum corneum was significantly decreased from 30 min until day 2, whereas that in the lower layer remained normal. In contrast, the amount of active SCCE showed no change during the experimental period. The surface pH of the stratum corneum drastically decreased to pH 2 at 30 min and slightly recovered to around pH 3 until 1 day after treatment. From 9 to 19 days, a decrease in corneocyte cell area and a remarkable long-term increase in the amount of active CD in the upper layer were observed. In an in vitro study, the activities of desquamation-regulating proteinases were shown to have remarkably increased at around pH 3, due to activation of CD at its optimal pH. These results suggest that GA functions via at least two different mechanisms, acute activation of CD in the lower layer by acidification around pH 3, along with inactivation of CD in the upper layer, and long-term enhancement of de novo CD production in the few weeks following GA treatment.