Aims: The aim of this study is to improve exoinulinase production by expression of a cloned exoinulinase gene inuA1 (GenBank accession no. JF961344) from Penicillium janthinellum strain B01 in Pichia pastoris. Methods and Results: A full-length cDNA of exoinulinase gene (inuA1) was cloned from P. janthinellum strain B01 using RACE PCR. An open reading frame (ORF) of 2115 bp is interrupted by a single intron of 67 bp. The fragment encodes a signal peptide with 20 amino acids and a mature protein with 684 amino acids. The inuA1 was subcloned to the pPICZ alpha C expression vector and succesfully over-expressed in Pichia pastoris X-33. The highest activity of exoinlinase reached 272.8 U ml(-1) in the fermentation liquid. It was c. 11-fold of that produced by wild-strain B01. A large amount of fructose was identified after the hydrolysis of inulin with the crude recombinant exoinulinase. The recombinant exoinulinase was purified and characterized. The molecular weight of the purified recombinant exoinulianse was 100 kDa. The mass spectrometry result indicated that the purified protein was indeed recombinant exoinulinase. The optimal pH and temperature of the purified recombinant exoinulianse were 4 5 and 50 degrees C, respectively. Conclusions: An exoinulinase gene of P. janthinellum strain B01 was cloned, sequenced and over-expressed successfully in P. pastoris. Significance and Impact of the Study: Only a few genes have been cloned from P. janthinellum because its molecular biology is poorly understood. In this study, we cloned and over-expressed inuA1 gene of P. janthinellum in P. pastoris. This recombinant exoinulinase can be used to hydrolyse inulin to produce fructose and facilitate the biofuel production from inulin resources.