The cell surfaces of microorganisms display distinct molecular patterns formed from lipopolysaccharides, peptidoglycans, or beta1,3-glucans. Binding of these surfaces by pattern recognition proteins such as beta1,3-glucan recognition proteins (betaGRPs) activates the immune response in arthropods. We identified a 40-kDa beta1,3-glucan-binding protein with sequence similarity to previously characterized lepidopteran betaGRPs from hemolymph, but unlike these it is secreted into the larval gut lumen and is an active beta1,3-glucanase. This glucanase was not detected in hemolymph. Its mRNA is constitutively and predominantly expressed in the midgut and is induced there when larvae feed on a diet containing bacteria. Homologs of this predominantly midgut-expressed gene from many Lepidoptera possess key residues shown to be part of the active site of other glucanases, and form a cluster that is distinct from previously described betaGRPs. In addition, this group includes proteins from insects such as the Anopheles gambiae GNBP subgroup B for which a catalytic role has not been previously suspected. The current domain classification does not distinguish between the catalytic and noncatalytic clades, and should be revised. The noncatalytic betaGRPs may be evolutionarily derived from this newly described enzyme family that continues to function catalytically in digestion and/or pathogen defense.