Abstract: Ratooning rice, with significant improvement in potential yield, has become a major farming system to raise multiple crop index. Ratooning rice yield is directly related to the rate of ratooning bud germinating into spikes, and thereby enhancing ratooning bud germination is a key element for ratooning rice high yield. Numerous studies on rice eco-physiology have shown that nitrogen application enhances bud development also strongly promotes ratooning bud germination. However, the molecular mechanism of nitrogen application for bud development to promote ratooning bud germination has remained unclear. This paper studied the effects of nitrogen application for bud development on protein expression of ratooning buds of rice and the corresponding physiological characteristics by using comparative proteomics and relative physiological indicators. A total of 20 differentially expressed proteins were detected in different treatments in 2-DE maps of ratooning bud proteome. The proteins involved in energy metabolism, growth regulation, defense response to stress, etc. The results indicated that the expression abundance of nucleoside diphosphate kinase protein involved in energy metabolism was down-regulated and the proteins involved in photosynthesis (probable photosystem Ⅱ oxygen-evolving complex protein and cytochrome b6-f complex iron-sulfur subunit) were up-regulated during ratooning bud germination. Furthermore, protein expressions were higher under nitrogen application treatment than non-fertilization treatment on 7 d and 14 d after nitrogen application for bud development. It suggested that nitrogen application for ratooning bud development significantly slowed the down-regulation of proteins involved in energy metabolism, and promoted the up-regulation of proteins involved in photosynthesis. Thus ratooning buds mediated by fertilization treatment had relatively stronger energy synthesis and photoautotroph capabilities, which better met the needs of energy and material for ratooning bud germination and differentiation. Six protein spots involved in cellular growth and five protein spots involved in resistance were detected and identified in the study. Among them, two protein spots (translationally-controlled tumor protein homolog and actin-depolymerizing factor) were down-regulated and nine protein spots (e.g., germin-like protein, ABA/WDS-induced protein, putative quinone-oxidoreductase QR2, Cu-Zn superoxide dismutase and dirigent-like protein) were up-regulated during the ratooning bud germination. Furthermore, expression abundance of proteins (e.g., ABA/WDS-induced protein and actin-depolymerizing factor on the 7^th and 14^th day, germin-like protein on the 7^th day of fertilization) was lower under nitrogen application treatment than non-fertilization treatment. Proteins (e.g., translationally-controlled tumor protein homolog, putative quinone-oxidoreductase QR2, Cu-Zn superoxide dismutase and dirigent-like protein on the 7^th and 14^th day, germin-like protein on the 14^th day) had higher expressions under fertilization treatment than non-fertilization treatment. It indicated that nitrogen application for ratooning bud development relatively cut down the expression abundance of proteins that inhibited ratooning bud germination. Meanwhile, it increased the expression abundance of proteins related to cell division, elongation and resistance, which promoted the germination of ratooning bud and enhanced the ability to resist adverse conditions. Therefore compared with those under non-fertilization treatment, living bud rate and length of the 2nd to the 5^th ratooning bud were significantly higher in nitrogen application treatment. As a result, nitrogen application for bud development significantly improved effective panicle number, seed setting rate and grain yield of ratooning rice, which was aimed at getting high yield and significant improvement in ratooning rice production.