(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.