Technical Abstract: The present work characterized a second endogenous cellulase (endo-ß-1,4-glucanase) gene, CfEG4, uncovered in the transcriptome of Formosan subterranean termite (Coptotermes formosanus). The full-length gene was cloned and sequenced. It is similar to the CfEG3a described earlier (Zhang et al. 2009) but not likely an allelic variant. GenomeWalker™ DNA walking analysis indicated that there may be one copy of CfEG4 and two copies of CfEG3a in the termite genome. As with CfEG3a, the transcript of CfEG4 was detected predominantly in the salivary gland based on quantitative RT-PCR. Phylogenetic analysis of translated amino acid sequence showed that the CfEG4 is more related to CaEG, derived from an Australian subterranean termite (C. acinaciformis), than CfEG3a and other cellulases from C. formosanus, Reticulitermes speratus, or R. flavipes. Recombinant CfEG4, produced in E. coli, was active against filter-paper cellulose, resulting in mostly cellobiose and cellotriose, similar to the enzymatic and biochemical properties of CfEG3a. These findings would lead to further investigation of both the evolutionary origin of eukaryotic cellulase genes and the evolutionary relationship of termite species. The cellulose-degrading enzyme is applicable for bioconversion of wood to simple sugars and production of biofuels. The recombinant cellulase should also be useful for designing and screening of inhibitors for the development of target-specific and environment-friendly bio-termicides