Acquired enamel pellicle plays an important role in the pathogenesis of early childhood caries (ECC), working as a protective interface between the tooth and the oral cavity. The aim of this cross-sectional in vivo proteomic study was to compare the acquired enamel pellicle protein profile of 3-5-year-old children with ECC (n = 10) and caries-free children (n = 10). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). In total, 241 proteins were identified. Basic salivary proline-rich protein 1 and 2, Cystatin-B, and SA were found only in the caries free group. When comparing caries free and ECC groups, lower protein levels were found in the caries free group for hemoglobin subunit beta, delta, epsilon, gamma-2, globin domain-containing protein and gamma-1, neutrophil defensin 3, serum albumin, protein S100-A8, and S100-A9. The proteins histatin-1, statherin, salivary acidic proline-rich phosphoprotein ½, proline-rich protein 4, submaxillary gland androgen-regulated protein 3B, alpha-amylase 1 and 2B were found at higher levels in the caries free group. The exclusive and the proteins found at higher levels in the caries free group might have protective functions that play a role in the prevention of caries, besides providing important insights to be evaluated in future studies for the possible development of new therapeutic strategies for ECC.