Background: Alopecia is a hair loss disorder, which can have a significant psychological impact on the quality of life. Silibinin is a secondary metabolite from Silybum marianum, which is a major active component of silymarin. Previous studies have revealed that silibinin acts as an antioxidant, which can reduce the risk of cutaneous cancers. Objectives: This study aimed to evaluate the effect of silibinin on hair growth in 3-dimensional (3D) cultured human dermal papilla (DP) spheroids. Methods: Activation of serine/threonine kinase (Akt) and the wingless and INT-1 (Wnt)/β-catenin signaling pathway were measured by using the western blotting and the T cell-specific transcription factor and lymphoid enhancer-binding factor (TCF/LEF) promoter-carrying luciferase reporter assay. Expression levels of genes were determined by using quantitative real time PCR analysis. Results: Silibinin significantly increased cell viability through Akt activation in 3D-cultured DP cells. After the treatment with silibinin, the Wnt/β-catenin signaling pathway was highly activated, and the expression levels of the target genes, WNT5a and LEF1, were increased in 3D DP spheroids. Moreover, the expression levels of the signature genes of DP, such as ALPL, VCAN, FGF7 and BMP2, were significantly increased. Conclusion: Silibinin induces hair growth in 3D-cultured DP spheroids through the Akt and Wnt/β-catenin signaling pathways. This study suggests that silibinin can be a potential therapeutic option for alopecia.