Abstract: Liquid culture method and mycelial growth rate method were performed to explore the antifungal activity of Bacillus sp. CmRh1 fermentation broth induced by Agar buffered Pachyman (ABP) medium against Colletotrichum gloeosporioides in this study. The endoglucanase gene was cloned by polymerase chain reaction (PCR) technique, heterologously expressed in Escheria coli and functionally analyzed. Physicochemical properties, signal peptide, transmembrane region, conserved domain, secondary and tertiary structure, and subcellular location of endoglucanase from Bacillus sp. CmRh1 were predicted by the online bioinformatics tools. The results showed that Bacillus sp. CmRh1 possessed the ability to produce β-1,3 glucanase and β-1,4 glucanase, and the fermentation broth containing glucanase displayed a certain antifungal activity against C. gloeosporioides mycelial growth. Besides, an endoglucanase gene (Glu4) from Bacillus sp. CmRh1 was cloned, and its open reading frame (ORF) was 1500 bp encoding a protein of 499 amino acid residues. Bioinformatics predicted that Glu4 was a stable hydrophilic protein containing a signal peptide and a transmembrane domain. The relative molecular weight of Glu4 was 55 kDa, and its theoretical isoelectric point was 7.14. Glu4 belonged to the glycoside hydrolase family (GH5) containing the carbohydrate-binding module (CBM_3). The secondary structural elements mainly include α helix, β turn, extended strand, and random coil. The tertiary structure was a typical β-sandwich structure, which was consistent with the structural characteristics of endoglucanase. It was predicted that Glu4 was located outside the cell. The results of combined SDS-PAGE with western blot showed that Glu4 was successfully expressed in E. coli, and its molecular weight was consistent with the prediction. In conclusion, E. coli BL21(DE3)/pEB-Glu4 had the ability to secrete endoglucanase, which would be beneficial to purifying, analyzing characterization of endoglucanase, and exploring antifungal activity of endoglucanase against C. gloeosporioides.