This study investigated an endoglucanase (EGII) from Rhizopus stolonifer var. reflexus TP-02 that consists of a C-terminal catalytic domain and an N-terminal carbohydrate-binding module joined by a linker rich in glycine, serine, threonine, and alanine. Site-directed mutagenesis was applied to characterize the conformation and dynamics of the linker. Mutants were expressed in Escherichia coli BL21 and purified by Ni-chelating column. Structural analysis indicated that glycine provided flexibility in the enzymatic process. G67P, G91Y, G101Y, G108Y, G109Y, G112P, H61G, H75G, and Y103G were selected on the basis of the results of the bioinformatics and Ramachandran plot analysis for the linker. The catalytic activities of EGII and its mutants on CMC-Na, microcrystalline cellulose (Avicel), and phosphoric acid-swollen celluloses (PASC) showed that flexible amino acids strengthened the activity of the enzyme. It indicated that flexible amino acids could improve the flexibility of the linker. Overall, the linker affected the catalytic efficiency of the endoglucanase in hydrolyzing cellulose chains.