Different from humans, who have a continuous dentition of teeth, mice have only three molars and one incisor separated by a toothless region called the diastema in the hemi mandibular arch. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. In this study, we evaluated the proteins that modulate the diastema formation through comparative analysis with molar-forming tissue by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteome analysis. From the comparative and semi-quantitative proteome analysis, we identified 147 up- and 173 down-regulated proteins in the diastema compared to the molar forming proteins. Based on this proteome analysis, we selected and evaluated two candidate proteins, EMERIN and RAB7A, as diastema tissue specific markers. This study provides the first list of proteins that were detected in the mouse embryonic diastema region, which will be useful to understand the mechanisms of tooth development.