Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme's commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis. Mutants were constitutively expressed in Pichia pastoris, purified, and ultimately tested for enzymatic characteristics. Two single mutants, Y30F and Y173F, increased the enzyme's specific activity 1.35- and 1.87-fold using carboxymethylcellulose sodium (CMC-Na) as a substrate, respectively. Furthermore, CTendo45 and mutants exhibited higher activity towards β-D-glucan than that of CMC-Na, and activities of Y173F and Y30F were also increased obviously against β-D-glucan. In addition, Y173F significantly improved the enzyme's heat resistance at 80 °C and 90 °C. More interestingly, the double mutant Y30F/Y173F obtained considerably higher stability at elevated temperatures but failed to inherit the increased catalytic efficiency of its single mutant counterparts. This work gives an initial insight into the biological function of conserved and noncatalytic residues of thermostable endoglucanases and proposes a feasible path for the improvement of enzyme redesign proposals.