Abstract: Brassica napus L., the predominant variant of rapeseed cultivated in China, undergoes a series of complex metabolic and regulatory processes during flower bud development, especially the early developmental stages preceding microspore maturation, which significantly impacts fertility. Based on phenotypic and histological observations, flower bud sizes ranging from 1 mm to 3 mm in B. napus were found to correspond to the 5^th–9^th stages of angiosperm stamen development. Using high-throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) sequencing technology, proteomics analysis was conducted on these flower buds. In total, 13 444 proteins were identified, including 613 differentially expressed proteins (DEPs). Functional analysis of the DEPs revealed a significant up-regulation in cell wall building, lipid metabolism, and cell transport-related proteins, crucial for cell development and pollen formation during the early stage of flower bud development. The expression of nucleic acid-related proteins markedly changed, consistent with rapid cell division and growth during this period. However, no significant changes were found in the expression of proteins related to the ABCDE development model, suggesting these proteins may have other functions beyond inducing flower bud differentiation. This study systematically studied the differences in tissue morphology and protein expression during the early stages of bud development in B. napus, paving the way for further research on the metabolism and regulation of flower bud development.