The cDNA of a human Mini-proinsulin (B¹-B²⁹)-AAK-(A¹-A²¹) which named HMPIDesB³⁰ (human mini-proinsulin des B³⁰) was synthesized and inserted into the Escherichia coli-yeast shuttle vector pPIC9K. The constructed plasmid, HMPIDesB³⁰/pPIC9K was transformed into the GS115 cells of the methylotrophic yeast, Pichia pastoris, using electrotransformation. A transformant with a high copy number of the gene integrated into the chromosome was obtained by YPD plates which contains increasing concentrations of G418. After fed-batch fermentation, the protein was purified by macroporous resin, ion-exchange chromatography and precipitated with 10～20 mmol/L Zn²⁺. The purity of HMPIDesB³⁰ is 95%，the expression level reached 1.0 g/L, and the recovery rate of purification is about 60%. The purified protein was characterized by 16.5% Tricine SDS-PAGE, HPLC, and mass spectrometry, and the molecular mass of the expressed products is in accordance with the cumulated value. HMPIDesB³⁰ can be secretorily expressed by Pichia pastrois. The composition of the fermentation medium, the optimal condition of the fermentation and an effective method to purify the expression products from the culture were established.