beta-1,3-glucanases are the main digestive enzymes of plant and fungal cell wall. Transcriptomic analysis of the fungus-growing termiteMacrotermes barneyirevealed a high expression of a predicted beta-1,3(4)-glucanase (Mbbgl) transcript in termite gut. Here, we described the cDNA cloning, heterologous expression, and enzyme characterization of Mbbgl. Sequence analysis and RT-PCR results showed that Mbbgl is a termite-origin GH16 beta-1,3(4)-glucanase. The recombinant enzyme showed the highest activity towards laminarin and was active optimally at 50 degrees C, pH 5.5. The enzyme displayed endo/exo beta-1,3(4)-glucanase activities. Moreover, Mbbgl had weak transglycosylation activity. The results indicate that Mbbgl is an endogenous digestive beta-1,3(4)-glucanase, which contributes to the decomposition of plant biomass and fungal hyphae. Additionally, the multiple activities, pH, and ion stabilities make Mbbgl a potential candidate for application in the food industry.