Phosphoglucose isomerase (PGI) is involved in synthesizing extracellular polysaccharide (EPS). The gene encoding PGI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli, and the protein was characterized. The pgi gene from DJ77 is 1,503 nucleotides long with 62% GC content and the deduced amino acid sequence shows strong homology with PGIs from other sources. The molecular masses of PGI subunit and native form were estimated to be 50 kDa and 97 kDa, respectively. Four potentially important residues (H361, R245, E330 and K472) were identified by homology modeling. The mutations, H361A, R245A, E330A, R245K and E330D resulted in decrease in Vmax by hundreds fold, however no significant change in Km was observed. These data suggest that the three residues (H361, R245Aand E330) are likely located in the active site and the size as well as the spatial position of side chains of R245 and E330 are crucial for catalysis.