A mutant human thrombopoietin (TPO) gene with a modified translation initiation region (TIR) sequence was created by site-specific mutagenesis based on the PCR technique. This mutant TPO gene encoded the same amino acid sequence as wild-type TPO gene. The wild-type TPO gene was expressed in E. coli with very low efficiency. The mutant TPO gene could reach an expression level of up to 10% of total cellular proteins in E. coli, which was much higher than the wild-type gene. The recombinant protein was mainly in the form of inclusion body which could acquire in vitro activities of human thrombopoietin after refolding.