The pulA1 gene, encoding a novel thermostable type I pullulanase PulA1 from Bacillus sp. CICIM 263, was identified from genomic DNA. The open reading frame of the pulA1 gene was 2655 base pairs long and encoded a polypeptide (PulA1) of 885 amino acids with a calculated molecular mass of 100,887 Da. The pulA1 gene was expressed in Escherichia coli and Bacillus subtilis. Recombinant PuLA1 showed optimal activity at pH 6.5 and 70 °C. The enzyme demonstrated moderate thermostability as PuLA1 maintained more than 88% of its acitivity when incubated at 70 °C for 1 h. The enzyme could completely hydrolyze pullulan to maltotriose, and hydrolytic activity was also detected with glycogen, starch and amylopection, but not with amylose, which is consistent with the property of type I pullulanase. PulA1 may be suitable for industrial applications to improve the yields of fermentable sugars for bioethanol production.