A DNA fragment encoding dextranase was cloned from Penicillium pinophilum SMCU3-14 using the genome walking approach. Sequence analysis of the gene (SMCU-DEX) revealed a putative CAAT box at −165 (CCAAT), a putative TATA box at −93 (TATAA) in the 5 -noncoding region, and a polyadenylation signal (AATAAG) in the 3 -noncoding region. A cDNA sequence analysis revealed no evidence of introns. The deduced open reading frame is 1824 bp in length and encodes a predicted protein of 608 amino acids (molecular weight (MW) of ∼66 kDa), with a putative N-terminal 20-amino acid signal peptide, giving a predicted mature protein of 588 amino acids (MW of ∼64 kDa) that belongs to glycosyl hydrolase family 49, as with other fungal dextranases. This is the first report of a dextranase gene sequence from P. pinophilum. The cDNA was cloned and expressed in Escherichia coli, and the transformants showed dextranase activity on a dextran-containing agar medium. Crude extracts from the transformants analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis containing blue dextran revealed a distinct specific band of dextranase activity at an MW of approximately 66 kDa. This recombinant dextranase is likely to have valuable and cost-effective applications in medicine and industry.