The expression of a pullulanase gene in Pichia pastoris was investigated. The gene encoding pullulanase was cloned by PCR using the chromosomal DNA of Bacillus naganoensis as the template. The expression vector pPIC9K-Pu was constructed by inserting the pullulanase gene into plasmid pPIC9K and then transformed into Pichia pastoris SMD 1168 by electroporation. Activity determination, SDS-PAGE, and PCR amplification indicated that the gene of the pullulanase from B. naganoensis had successfully been expressed in SMD 1168 and the molecular size of the expressed recombinant product was about 119.9 kDa. This is the first report on the successful expression of the pullulanase from B. naganoensis in P. pastoris. The transformant secreted recombinant pullulanase with the activity of 350.8 IU/ml in shake-flask culture. The properties of the recombinant pullulanase were characterized.