Popcorn is a special kind of maize that can easily become popcorn flake when heated under a given condition. However, its popping mechanism remains unclear at present. Opaque2 (O2) was a central transcriptional factor that play important roles during maize endosperm development. In this study, an o2 introgression into N04 inbred line was constructed, and the mutation of o2 reduced the popping rate of N04. In order to reveal the underlying mechanism of O2 involved in popping characteristics, the o2 introgression and popcorn inbred line N04 were performed proteomic analysis at three development stage of their kernels using the 8-plex iTRAQ reagents with three repetitions followed by 2D-LC-MS/MS spectra. A total of 5184 proteins were found, and 1073 proteins of which showed dynamics change. KEGG enrichment analysis found that these dynamic changed proteins were significantly enriched in carbon metabolism, pyruvate metabolism, citrate cycle, fructose and mannose metabolism, starch and sucrose metabolism, and pentose phosphate pathway. A total of 16 differentially expressed protein regulated by O2 were identified. These results preliminary uncover the molecular mechanism of popping characteristics in popcorn.