Abstract: Aiming to improve the fermention yield of insulin precursor (IP) in Pichia pastoris, a spacer peptide of 10 amino acids (EEAEAEAEPK) was introduced into the N terminal of the IP. This sequence might cause the possibility of the N terminal heterogeneity of the products. The IP produced in Pichia pastoris was purified by ion exchange chromatography and reversed phase chromatography. The purified samples were analyzed with MALDI TOF MS and N terminal sequencing. Results proved the existence of N terminal heterogeneity and helped to find the possible causes for this phenomenon. The results also indicated that the IP produced by P. pastoris could generate a mutated insulin with deletion of threonineB30(desB30) in chain B which posessed the characteristics of N terminal homogeneity and no degradation in C terminal after digested by trypsin. This research provided a new method for human insulin and detemir insulin production.