Activation of the Wnt/β-catenin signaling pathway plays an important role in hair follicle morphogenesis and hair growth. Recently, low-level laser therapy (LLLT) was evaluated for stimulating hair growth in numerous clinical studies, in which 655-nm red light was found to be most effective and practical for stimulating hair growth. We evaluated whether 655-nm red light + light-emitting diode (LED) could promote human hair growth by activating Wnt/β-catenin signaling. An in vitro culture of human hair follicles (HFs) was irradiated with different intensities of 655-nm red light + LED, 21 h7 (an inhibitor of β-catenin), or both. Immunofluorescence staining was performed to assess the expression of β-catenin, GSK3β, p-GSK3β, and Lef1 in the Wnt/β-catenin signaling. The 655-nm red light + LED not only enhanced hair shaft elongation, but also reduced catagen transition in human hair follicle organ culture, with the greatest effectiveness observed at 5 min (0.839 J/cm2). Additionally, 655-nm red light + LED enhanced the expression of β-catenin, p-GSK3β, and Lef1, signaling molecules of the Wnt/β-catenin pathway, in the hair matrix. Activation of Wnt/β-catenin signaling is involved in hair growth-promoting effect of 655-nm red light and LED in vitro and therefore may serve as an alternative therapeutic option for alopecia.