OBJECTIVE This in situ study aimed to evaluate the protective effect of Tooth Mousse (GC) containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on human enamel erosion and to compare the difference in erosion between the anteriorly and posteriorly positioned human enamel. METHODS This study used a 2-treatment (7 days each) crossover design with 12 healthy volunteers wearing intraoral appliances. Each appliance contained 4 human enamel specimens positioned on the buccal surfaces of the volunteers' maxillary central incisors and first molars. The specimens were intraorally treated withTooth Mousse (CPP-ACP group) or deionized water (control group) for 3 min and then exposed to in vivo acid attacks by rinsing with 150 ml of a cola drink (4 × 5 min/day). The surface microhardness (SMH) of the specimens was measured and used to calculate the percentage of SMH loss (%SMH). Erosion effect on enamel was also investigated by scanning electron microscopy (n = 4) at the end of study. The data were statistically analysed using two-way analysis of variance (ANOVA) and Tukey's test at a level of P < 0.05. RESULTS A significant decrease in %SMH was observed for the specimens of CPP-ACP group compared to that for the controls (P = 0.007). The specimens positioned posteriorly exhibited a significantly lower %SMH than those positioned anteriorly (P = 0.033). Samples of CPP-ACP group showed fewer etching patterns than those of the control group. CONCLUSIONS In this in situ model, application of Tooth Mousse containing CPP-ACP before erosion reduced the %SMH of human enamel. Enamel located in different positions showed different patterns of erosion. CLINICAL SIGNIFICANCE Application of Tooth Mousse containing CPP-ACP could be considered as a suitable preventive strategy against enamel erosion. ClinicalTrials.gov Identifier: NCT03426150.