This study utilized the Pichia pastoris expression system for expression of the synthetic human calcitonin (hCT) gene, a small peptide hormone secreted by the thyroid gland in mammals and ultimobranchial glands in lower vertebrate. The P. pastoris vector (pPICZB) contains the alcohol oxidase gene promoter (AOX1), which under the induction of methanol allows for the expression of heterologous protein gene inserted downstream in the vector. KM71H (muts ) strain of P. pastoris was used as the host cell. Molecular analysis, including polymerase chain reaction (PCR), sequencing, restriction enzyme analysis and survival of P. pastoris to increase concentration of zeocin antibiotic showed that human calcitonin gene was successfully integrated into the P. pastoris genome. The expected peptide which had an apparent molecular mass of 5.5 kDa was detected by Tricine-SDS-PAGE analysis and confirmed by enzyme-linked immunosorbent assay (ELISA).