A xyloglucanase (xyloglucan-specific endo-β-1,4-glucanase [EC 3.2.1.151]), AoXEG29, was purified from Aspergillus oryzae RIB40 to 75.5-fold of its apparent homogeneity with 1.2% recovery. It was a monomeric protein with a molecular mass of approximately 29 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration and zymogram analysis. Sugar-chain staining showed a sugar chain in the purified enzyme. AoXEG29 hydrolyzed detectable amounts of xyloglucan and AZCL-xyloglucan, but not CMC, H₃PO₄-swollen cellulose, oat-spelt xylan, low-melting-point agarose, glucomannan, colloidal chitin, or AZCL-xylan. The optimum pH at 40°C and optimum temperature at pH 5.0 were 4.5-5.5 and 60°C, respectively. AoXEG29 was stable at pH 3-8 at 40°C for 30 min and up to 50°C at pH 5 for 20 min. The kinetic parameters K _m and V _max for the hydrolysis of tamarind xyloglucan were 2.4 mg/mL and 1,250 U/mg, respectively.