To establish proteomic maps of Bifidobacterium longum subsp. longum BBMN68, applying two-dimensional gel electrophoresis, we built a 2-D reference map, by MALDI-TOF mass spectrometry. We identified a total number of 206 different proteins (11.4% of predicted total proteins in B. longum subsp. longum BBMN68). A total of 800±15 (exponential) and 800±20 (stationary) protein spots on each 2D–PAGE map were got, in which a total of 282 spots were successfully identified, represented 206 different proteins. Additionally, combined with analysis of pI and molecular masses, COG, CAI, GRVAVY and the cellular localizations of identified proteins in B. longum subsp. longum BBMN68. This study presented a proteome map of B. longum BBMN68 and provided a reference proteome database for comparative proteomic studied.