β-glucosidase from the midgut of the fungus-growing termite Macrotermes barneyi was first cloned and characterized to gain a better understanding of cellulolytic systems in fungus-growing termites. β-glucosidase activity was proven to present primarily in the midgut of M. barneyi and two β-glucosidases were partially purified from the midgut. Based on the N-terminus sequence of one of the β-glucosidases, a full-length cDNA fragment of 1708 bp was obtained. This sequence encodes a 493 amino acid protein belonging to glycoside hydrolase family 1. Quantitative real-time PCR analysis proved that the β-glucosidase gene was primarily expressed in the midgut. β-glucosidase was expressed heterologously and biochemically characterized. Results indicate that β-glucosidase is an endogenous, midgut-origin termite digestive enzyme. It may have applications in understanding the mechanism of lignocellulose degradation in fungus-growing termites.