Two cytosolic nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductases, Gox1899 and Gox2253, from Gluconobacter oxydans 621H were overproduced and purified from Escherichia coli. The purified proteins exhibited subunit masses of 26.4 (Gox1899) and 36.7 kDa (Gox2253). Both proteins formed homo-octamers exhibiting native masses of 210 and 280 kDa, respectively. The substrate spectra, optimal reaction conditions, and kinetic constants were determined for Gox1899 and Gox2253. Both enzymes efficiently catalyzed the reduction of medium/long-chain aldehydes. However, Gox1899 had a wider substrate spectrum and was more catalytically efficient. The best activity with Gox1899 was found for aliphatic aldehydes of C6-C10. In contrast, Gox2253 had a limited substrate spectrum and reduced octanal, nonanal, and decanal. Both enzymes were unable to oxidize primary alcohols. Aldehyde removal may be of particular importance for Gluconobacter because the membrane-bound alcohol dehydrogenase rapidly oxidizes short to long-chain alcohols, and large quantities of aldehydes could enter the cell, making detoxification necessary.