In the past two decades, accumulated evidence has clearly demonstrated the inhibitory effects of laser irradiation on enamel demineralization, but the exact mechanisms of these effects remain unclear. The purpose of this study was to investigate the effects of low-energy CO2 laser irradiation on demineralization of both normal human enamel and human enamel with its organic matrix removed. Twenty-four human molars were collected, cleaned, and cut into two halves. One half of each tooth was randomly selected and its lipid and protein content extracted. The other half of each tooth was used as the matched control. Each tooth half had two window areas. All the left windows were treated with a low-energy laser irradiation, whereas the right windows served as the non-laser controls. After caries-like lesion formation in a pH-cycling environment, microradiographs of tooth sections were taken for quantification of demineralization. The mean mineral losses (with standard deviation) of the enamel control, the lased enamel, the non-organic enamel control, and the lased non-organic enamel subgroups were 3955 (1191), 52(49), 4565(1311), and 1191 (940), respectively. A factorial ANOVA showed significant effects of laser irradiation (p = 0.0001), organic matrix (p = 0.0125), and the laser-organic matrix interaction (p = 0.0377). The laser irradiation resulted in a greater than 98% reduction in mineral loss, but the laser effect dropped to about 70% when the organic matrix in the enamel was removed. The results suggest that clinically applicable CO2 laser irradiation may cause an almost complete inhibition of enamel demineralization.