In this work, the high-level expression of the human lysozyme (HLY) was achieved by both optimization of the gene copy number and co-expression of the transcription factor Hac1p for the unfolded protein response (UPR) in the host strain Pichia pastoris KM71H. A series of recombinant constructs with various numbers of HLY expression cassettes was generated for the production of recombinant strains integrated with different copies of the HLY gene. The copy number of the HLY gene was determined by real-time quantitative polymerase chain reaction, and the recombinant strains of P. pastoris carrying one, two, three, four, or six copies of the HLY gene were obtained. Maximum extracellular protein and lysozyme enzyme activity reached 436.99 +/- 26.08 mu g/mL and 61,900 +/- 2036.47 U/mL, respectively, in the recombinant strain HLYH4-3 with the four copies of the HLY gene after shaking flask fermentation. Moreover, the co-expression of the transcription factor Hac1p in the recombinant strains further enhanced the HLY yields. Extracellular protein and lysozyme enzyme activity, respectively, reached 517.82 +/- 4.19 mu g/mL and 78,600 +/- 1134.95 U/mL by using the Hac1p co-expression strain HLYH4-3/Hac1p. These values are the highest recorded level of human lysozyme expressed by P. pastoris in shaking flask fermentation so far.