The proteome of mature (MP) and in vitro germinating pollen (GP) of canola (Brassica napus) were analyzed using the DIGE technology with the objective of identifying proteins and their function in pollen germination. Of the 2,238 protein spots detected in gel images, 344 were differentially expressed in MP and GP samples of which 165 were subjected to MALDI-TOF/TOF and 130 were successfully identified using the NCBInr and Brassica EST databases. The major proteins up-regulated in GP, relative to MP, have roles in carbohydrate metabolism, protein metabolism, and cell wall remodeling. Others with roles in cytoskeleton dynamics, nucleotide and amino acid metabolism, signal transduction, and stress response also showed higher expression in GP. Proteins concerned with transcriptional regulation and ion transport were similar in MP and GP, and some catalases and LEA proteins were down-regulated in GP. A number of proteins including, oleosin, cruciferin, and enolase, were released into the pollen germination medium indicating their potential role in pollen-stigma interaction. Glycosylated proteins were also identified in MP and GP, but their protein profiles were not different. This study has documented the dynamics of protein expression during pollen germination and early tube growth in B. napus and provides insights into the fundamental mechanisms involved in these processes, and in cell growth, cell-cell communication, and cell signaling.