Escherichia coli phytase appA, which hydrolyzes phytate, has been widely applied as an important feed supplement, but its resistance to trypsin needs to be improved. Six putative solvent-accessible amino acid residues (K74, K75, K180, R181, K183, and K363), which could be easily attacked by trypsin, were selected to improve trypsin tolerance of Escherichia coli phytase appA. Inspection of the three-dimensional structure and computational design via hydrogen bond analysis, six optimal mutation sites of K74D/K75Q/K180N/R181N/K183S/K363N, which strengthened the hydrogen bonding, were performed to generate three mutants. Results showed that the most beneficial mutant appA-M6 had a specific activity of 3262U/mg with molecular weight of approximately 52-55kDa. Similar to appA-WT, the optimal pH (4.5) and temperature (60 degrees C) of appA-M6 were unchanged. Compared with appA-WT, appA-M6 showed a significant enhancement (p<0.05) in resistance to trypsin and a 3.8 degrees C increase in melting temperature (T-m). We concluded that introduction of hydrogen bonds and N-glycosylation modification resulted in decreased enzyme flexibility and increased the enzyme stability against proteolysis and thermal denaturation. The mutant appA-M6 generated in this study could be applied for the large-scale commercial production of phytase and thus could benefit the food and feed industry.