An investigation of the structural differences between three newly determined cellobiose 2-epimerases (CEs) and other enzymes in the N-acyl-D-glucosamine 2-epimerase superfamily suggested that it was highly possible that His196 in Spirochaeta thermophila CE was involved in catalyzing isomerization. Rearrangement of the flexible loop during catalysis was significantly involved in the bifunction of this CE and the intermolecular interactions inside the flexible loop greatly influenced the reshaping of the loop and the catalytic direction.