Error-prone PCR was used to randomly mutate the endoglucanase (EG Ⅲ) gene of Trichoderma reesei followed by screening for clear-hole-forming colonies on carboxymethyl cellulose (CMC) plates at high pH. A mutant was selected with a shift of pH optimum from 4.8-5.0 to 5.4, and its specific enzyme activity decreased in low pH and increased in high pH. The sequencing of the mutant gene showed that the asparagine residue at 321 position was substituted by threonine. Two site-directed mutations, N321D and N321H, were designed to study the role of EG Ⅲ-321. It was discovered that the pH activity profiles of these two mutants had obvious change compared to the wild-type enzyme. It suggested that the residue at position 321 is a key amino acid residue in determining the pH-activity profile of the EG Ⅲ from T. reesei. The rule accorded with the alkali-tolerant mechanism of alkaliphilic cellulase K.