Background: Androgenetic alopecia (AGA) leads to thinning of scalp hair and affects 60%~70% of the adult population worldwide. Developing more effective treatments and studying its mechanism are of great significance. Previous clinical studies have revealed that hair growth is stimulated by 650-nm red light. Objective: This study aimed to explore the effect and mechanism of 650-nm red light on the treatment of AGA by using hair follicle culture. Methods: Human hair follicles were obtained from hair transplant patients with AGA. Hair follicles were cultured in Williams E medium and treated with or without 650-nm red light. Real-time RT-PCR and immunofluorescence staining were used to detect the expression level of genes and proteins in hair follicles, respectively. RNA-sequencing analysis was carried out to reveal the distinct gene signatures upon 650 nm treatment. Results: Low-level 650 nm red light promoted the proliferation of human hair follicles in the experimental cultured-tissue model. Consistently, 650 nm red light significantly delayed the transition of hair cycle from anagen to catagen . RNA-seq analysis and gene cluster- ing for the differentially expressed genes suggests that leukocyte transendothelial migration, metabolism, adherens junction and other biological process maybe involved in stimulation of hair follicles by 650-nm red light treatment. Conclusion: The effect of 650-nm red light on hair follicles and the transcriptome set which implicates the role of red light in promoting hair growth and reversing of miniaturiza- tion process of AGA were identified.