A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF MS and/or ESI–MS/MS. Isoelectric point values were estimated by gel electrophoresis, and molecular weight matched closely with their predicted values, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover, 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly, the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose, as determined by semi-quantitative RT-PCR. BL0033 time course and concentration experiments show that the induction time correlates to higher fructose concentration, and increased expression of BL0033. At the same time, an ABC transporter ATP binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared to glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system, in which BL0033 might play an important role.