Background: Many studies have indicated that hair growth and color regulation are cyclical processes involved in the strict regulation of multiple signaling pathways, such as the transforming growth factor and Wnt/β-catenin protein pathways. It has also been reported that Glycyrrhiza uralensis extract can promote hair growth, but its active components are still unclear. Objectives: The aim of this study was to investigate the potential hair blackening effect and mechanisms of glycyrrhetic acid (GA) extracted from Glycyrrhiza uralensis. Materials and Methods: Different concentrations of GA solution (1.0 wt%, 10.0 wt%, and 20.0 wt%) were coated onto the skin surface of fur model mice induced by H2O2 for two weeks. After that, the number and depth of follicles and cortex thickness were observed in the skin tissue stained by hematoxylin and eosin (H&E). The relative mRNA levels of β-catenin, vascular endothelial growth factor (VEGF), and tyrosinase in skin tissue were analyzed using quantitative real-time polymerase chain reaction. The protein levels of β-catenin, VEGF, and tyrosinase were also evaluated by western blotting and immunohistochemistry. Results: There was a marked increase in the number of hair follicles and the blackening effect upon exposure to 10.0 wt% and 20.0 wt% GA. Moreover, the β-catenin, VEGF, and tyrosinase expression levels in the skin tissue varied significantly compared to those in the model group. Conclusion: GA showed positive effects on hair growth factor expression levels in mice, which meant that GA could promote hair blackening.