Boron (B) deficiency is a worldwide problem, and Brassica napus is one of the most sensitive crops to B deficiency. To better understand the B starvation response of Brassica napus, we conducted a comparative proteomic analysis of seedling stage Brassica napus root between B-sufficient and B-limited conditions: 45 differentially expressed proteins were successfully identified by 2-DE coupled with MALDI-TOF/TOF-MS and LTQ-ESI-MS/MS analysis. Among these proteins, 10 were down-regulated and 35 were up-regulated under B-limited condition. Combining GO and KEGG analyses with data from previous reports, proteins were categorized into several functional groups, including antioxidant and detoxification, defense-related proteins, signaling and regulation, carbohydrate and energy metabolism, amino acid and fatty acid metabolism, protein translation and degradation, cell wall structure, and transporter. The genes of selected proteins were analyzed by quantitative RT-PCR. Our results provide novel information for better understanding the physiological and biochemical responses to B deficiency in plants.