Abstract: The embryo in high-oil maize is larger and has higher nutritional value. In this study, the embryonic proteome at 15 (G15), 25 (G25) and 35 (G35) days after pollination was isolated by two-dimensional electrophoresis in high-oil maize inbred line GY220, and the isolated proteins were identified by tandem mass spectrometry. The function of differentially expressed proteins were analyzed. The results showed that 41 differential proteins were identified in the three stages after pollination, among which 18 proteins were up-regulated expression and 19 were down-regulated expression by comparing G25 and G15, and 18 proteins upregulation and 25 down-regulation in G35/25, 7 proteins up-regulation and 22 down-regulation in G35/15. GO annotation analysis and KEGG enrichment analysis showed that the differential proteins were mainly enriched in small molecule metabolism, REDOX and carbohydrate metabolism. According to the transcriptomic database of inbred line B73 and RT-PCR analysis of differential protein encoding gene in inbred line GY220 showed that the expression of differential protein, glycerin-3-phosphate dehydrogenase and fructokinase, significantly increased in late embryo development, and they were highly homologous with related genes in soybean, peanut and rape. This study laid a foundation for further improving grain quality and mining functional genes of embryo development in maize.