We investigated the effects of CO2 laser irradiation combined with acidulated phosphate fluoride (APF) application on the demineralization of enamel. APF gel was applied to the buccal enamel of human premolars and CO2 laser was applied. After the specimens were immersed in demineralization solution for 72 h, they were subjected to depth-dependent micro-CT and nanoindentation analyses. Micro-X-ray diffraction and X-ray photoelectron spectroscopy were performed to analyze the surfaces. Some surface regions of the enamel in specimens that were laser-irradiated with low output and APF-treated showed significantly higher values of MD and hardness than specimens treated with APF alone. A higher fluoride concentration in the enamel surface was observed in specimens treated with CO2 laser irradiation plus APF gel application. In conclusion, CO2 laser irradiation with low output is preferable to improve acid resistance.