OBJECTIVES: This in situ study aimed to evaluate the effect of a new toothpaste formulation containing fluoride (F), casein phosphopeptide amorphous calcium phosphate (CPP-ACP) and sodium trimetaphosphate (TMP) on the remineralization of initial caries lesions. METHODS: This was a blind, crossover study, performed in 5 experimental phases with a duration of 3 days each. At first, the initial surface hardness (SH) and hardness after demineralization (SH RESULTS: The enamel surface showed greater remineralization (SH CONCLUSION: The result of this study demonstrates that the combined use of remineralizing agents (F, CPP-ACP, and TMP) in a single toothpaste formulation (1100F-CPP-ACP-TMP) significantly enhances mineral gain compared to conventional toothpaste, as evidenced by a short-term in situ model. CLINICAL SIGNIFICANCE: The findings of our study offer essential insights for future investigations and potential clinical applications, especially for individuals with initial caries lesions.