A naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were β-D-glucosidases and that the smallest subunit was an α-L-rhamnosidase. The naringinase and its α-L-rhamnosidase and β-D-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C, and they were stable between pH 3 and 6 and below 50 °C. This naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a K(m) value of 0.11 mM and a k(cat)/K(m) ratio of 14,034 s(-1) mM(-1) for total naringinase. Its α-L-rhamnosidase and β-D-glucosidase subunits had K(m) values of 0.23 and 0.53 mM, respectively, and k(cat)/K(m) ratios of 14,146 and 7733 s(-1) mM(-1), respectively. These results provide in-depth insight into the structure of the naringinase complex and the hydrolyses of naringin and other glycosides.