Objective: To evaluate the effects of casein phosphopeptide-amorphic calcium phosphate (CPP-ACP) on enamel erosion using an improved in situ experimental protocol. Methods: Forty-eight enamel blocks were prepared from fresh-extracted human premolars and further embedded in the acrylic resins. The present study was divided into 2 parts. In part 1 of the study, two volunteers were recruited to test the availability and safety of the in situ erosion protocol. Customized intraoral appliance was made with 4 reservoirs containing the specimens for each volunteer. For each intraoral applicance, 2 reservoirs were made with the openings and the other 2 remained intact. All volunteers were instructed to drink 150 ml cola within 5 min using the gargling method after placing appliances intraorally for 2 h. After erosion, the appliances were remained undisturbed intraorally for 1 h until the next erosive attack. The in situ erosion cycles were performed 4 times daily over 7 d. In part 2 of the study, forty specimens were randomly divided into 2 groups (n=20): CPP-ACP group and control group. The surfaces of specimens in CPP-ACP group were pretreated with CPP-ACP for 3 min before in situ erosion, whereas the specimens in control group were pretreated with deionized water for 3 min. For each intraoral applicance, 4 reservoirs were made with openings. Ten healthy volunteers were recruited and the above-mentioned in situ erosion protocol was applied to test the effects of CPP-ACP on enamel erosion. The surface microhardness and surface microstructure of the samples were examined before and after erosion in both parts of the study. The data were statistically analyzed using ANOVA and LSD tests. Data were considered statistically significant at a level of P<0.05. Results: Significant surface softening was observed in all specimens after erosion (P<0.001). The surface microhardness in the CPP-ACP group and control group were (198.8±23.2) and (152.4±42.1) HV, respectively (P=0.040). The specimens in the CPP-ACP group showed significantly fewer changes in surface microstructure compared with those in the control group. Conclusions: Based on this in situ experimental protocol, short-time consuming of acid beverages would produce significant effects on the surface microhardness of the human enamel, whereas the application of CPP-ACP can increase the erosion resistance of the enamel.