The airway epithelium is the footstone to maintain the structure and functions of lung, in which resident basal cells (BCs) maintain homeostasis and functional regeneration of epithelial barrier in response to injury. In recent clinical researches, transplanting BCs has shown great inspiring achievements in therapy of various lung diseases. In this study, we report a noninvasive optical method to activate BCs for airway epithelium regeneration in vivo by fast scanning of focused femtosecond laser on BCs of airway epithelium to active Ca2+ signaling and subsequent ERK and Wnt pathways. The photoactivated BCs present high proliferative capacity and maintain high pluripotency, which enables them to plant in the injured airway epithelium and differentiate to club cells for regeneration of epithelium. This optical method can also work in situ to activate localized BCs in airway tissue. Therefore, our results provide a powerful technology for noninvasive BC activation in stem-cell therapy of lung diseases.