Background: Improvement of microcirculation is one of the important mechanisms of low-level laser therapy (LLLT) to treat some diseases such as wound healing. Most previous studies have been carried out with multiple lasers other than the 10,600-nm CO2 laser. Recently, the CO2 laser has been used not only as a tool for excision of soft tissues but also for therapeutic applications. Objective: To study whether low-level CO2 laser irradiation can influence microcirculation and further explore the underlying mechanisms. Methods: Seventy-milliwatt (70-mW) CO2 lasers irradiated the forearms of 12 participants and skin blood perfusion (SkBP) was measured with a laser speckle imager. The thermal effect of irradiation was evaluated by measuring the irradiated skin in vivo and the exposed cell suspensions in vitro. Extracellular adenosine triphosphate (eATP) of the human mast cell line (HMC-1) is assessed by luciferin-luciferase assay to explore the potential mechanisms.