This study investigated the combined effect of CO2 laser irradiation and Remin Pro paste on microhardness of enamel white spot lesions (WSLs). Seventy-eight intact premolars were randomly assigned into six groups and then stored in a demineralizing solution to create WSLs. Afterwards, the teeth in group 6 (negative control) remained untreated, while groups 1 and 4 were exposed to CO2 laser irradiation (20 Hz, 1 W, 30 s) and Remin Pro paste, respectively. In groups 2 and 3, the teeth were exposed to laser either before (group 2) or after (group 3) Remin Pro application. The teeth in groups 1 to 5 were then immersed in artificial saliva for 90 days while subjected to fluoride mouthwash and weekly brushing. Finally, the teeth were sectioned, and Vickers microhardness was measured at the enamel surface and at 50, 100, and 150 μm from the surface. One sample of each group was also examined with scanning electron microscope (SEM). Data were analyzed by two-way analysis of variance (ANOVA) and Tukey's test. The significance was set at 0.05. Laser irradiation followed by Remin Pro application (group 2) caused a significant increase in total WSLs' microhardness compared with laser alone (group 1) and control groups (P < 0.05). Microhardness at depths of 100 and 150 μm was also significantly greater in group 2 compared with those of group 3 and control groups (P < 0.05). Combined application of CO2 laser with Remin Pro paste, when laser is irradiated before the paste, is suggested for re-hardening of WSLs in deep layers of enamel.