We have cloned a novel gene for d-sorbitol dehydrogenase (SLDH), which efficiently converted D-sorbitol to L-sorbose, from Gluconobacter oxydans G624 (FERM BP-4415). A cosmid library of the genomic DNA was screened by assaying SLDH activity. The inserted DNA from a positive clone was downsized by subcloning into charomid and pUCP plasmid, successively. Sequencing analysis of the DNA responsible for SLDH activity revealed an open reading frame of 1455 bp coding for 485 amino acid residues with a calculated molecular mass of 53,642 Da. The amino acid sequence showed 42.2% identity with a NAD+-dependent mannitol dehydrogenase (MDH), which catalyzed conversion of d-sorbitol to d-fructose, from Pseudomonas fluorescens DSM50106. Since the intact SLDH was found to be very unstable during isolation and purification, this SLDH fused to 6 x His-tag was expressed in Pseudomonas putida IFO3738 and purified by immobilized metal affinity chromatography using cobalt-based resins. The 6 x His-tag SLDH catalyzed the oxidation of D-sorbitol to L-sorbose and exhibited 15 times higher activity in the presence of NADP+ than that of NAD+. These results indicate that the SLDH is a novel kind of dehydrogenase distinct from MDH previously reported.