ABSTRACT Four types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designated bglA13 , bglA16 , bglA51 , and bglM2 , were found in the cDNA library of Neocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias to Streptococcus equinus . The presence of expansion and several predicted secondary structures in the 3′ untranslated regions (3′UTRs) of bglA16 and bglM2 suggest that these two genes were duplicated recently, whereas bglA13 and bglA16 , which contain very short 3′UTRs, were replicated earlier. These findings indicate that the β-glucanase genes from N. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes of Streptococcus equinus , Ruminococcus albus 7, and N. patriciarum J11 were cloned and expressed by Escherichia coli . The recombinant β-glucanases cloned from S. equinus , R. albus 7, and N. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higher k cat values than others. These results support the notion that duplicated β-glucanase genes, namely, bglA16 and bglM2 , increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms in N. patriciarum J11.