Objectives Hair loss, including alopecia, is a common dermatological issue worldwide. At present, the application of fractional carbon dioxide (CO2) laser in the treatment of alopecia has been documented; however, the results vary between reports. These varying results may be due to the limited knowledge of cellular action in laser‐irradiated skin. The objective of this study was to investigate the molecular and cellular mechanisms of laser treatment under effective conditions for hair cycle initiation. Methods A fractional CO2 laser was applied and optimized to initiate the hair cycle in a mouse model of alopecia. Several cellular markers were analyzed in the irradiated skin using immunofluorescence staining. Cellular populations and their comprehensive gene expression were analyzed using single‐cell RNA sequencing and bioinformatics. Results The effective irradiation condition for initiating the hair cycle was found to be 15 mJ energy/spot, which generates approximately 500 μm depth columns, but does not penetrate the dermis, only reaching approximately 1 spot/mm2. The proportion of macrophage clusters significantly increased upon irradiation, whereas the proportion of fibroblast clusters decreased. The macrophages strongly expressed C–C chemokine receptor type 2 (Ccr2), which is known to be a key signal for injury‐induced hair growth. Conclusions We found that fractional CO2 laser irradiation recruited Ccr2 positive macrophages, and induced hair regrowth in a mouse alopecia model. These findings may contribute to the development of stable and effective fractional laser irradiation conditions for human alopecia treatment.