Aims: This study attempted to fully explore the expression potentials of Pichia pastoris for producing porcine insulin precursor (PIP) through PIP copy number optimization. Methods and Results: Multi-copy strains were screened employing a highly efficient improved in vivo method and their copy numbers were quantified by real-time qPCR. A range of Mut(+)P. pastoris strains harbouring 0, 1, 3, 6, 12, 18, 29, 52 copies of PIP were obtained. After 96 h methanol induction, a bell-shaped correlation curve was observed between gene dosage and protein yield, and the maximum PIP expression level of 181 mg l(-1) was achieved by a 12-copy strain. Specific growth rate and methanol utilization capacity were found to decrease remarkably for high copy strains (> 12 copies). Transcriptional analysis of KAR2 suggested higher copy strains were suffering more from ER stress. Conclusions: A copy number around 12 is optimal for secretory expression of PIP in P. pastoris. Excess PIP gene dosage (> 12 copies) significantly impaired the growth of P. pastoris hosts. Significance and Impact of the Study: The methods developed and the discoveries made by this systematical investigation will be helpful to the application and understanding of Pichia pastoris expression system for heterologous overexpression.