Abstract: [Objective] Based on a proteomic reference map of the important probiotic organism Bifidobacteria longum NCC2705 constructed by our previous research, we compared the proteomic profiles of Bifidobacteria longum strain NCC2705 grown on lactose or glucose to identify the catabolic route allowing lactose fermentation. [Methods] We con-sidered the proteins differentially expressed if their relative volume deviated more than 3-fold with ImageMaster 2D Elite version 5.0 software. Interesting spots were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and phosphorylation analysis of proteins with mobility changes by Pro-Q Diamond Stain. [Results] The identified spots represent 31 protein entries, 14 up-regulated proteins, 17 down-regulated proteins. These identified proteins, which were hydrophilic proteins and their genes with CAI value above 0.5 represented the most abundant proteins, included key stress proteins, metabolism-related proteins, and proteins related to translation. Two proteins including Tal (BL0715, transaldolase, L3) and Pyk (BL0988, pyruvate kinase, G9) exhibited clear post-translational modification. [Conclusion] Proteomic comparison of glucose- and lactose-grown cells revealed that lactose and glucose were catabolized via the same degradation pathway, and the rate of glucose assimilation was higher than that of lactose. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Pro-Q Diamond staining analysis revealed that lactose trigger Tal phosphorylation at 43 T /47 S, and inhibited Pyk phosphorylation at 65 S. These proteins were identified for the first time as bifidobacterial phosphoproteins.