OBJECTIVE To construct tetracycline operator (TetO) modified glycophorin binding protein 130 gene (GBP130) promoter of Plasmodium falciparum and investigate the position effect of insertion of TetO on the promoter activity. METHODS Cloning of 7-copy of TetO (7cot) sequence into 4 points relative to transcriptional initiation site of GBP130 promoter in pGBPCATdelta2 plasmid (2 upstream and 2 downstream), respectively, produced 4 derivative plasmids, pG/7T (-5), pG/7T (-2), pG/7T (+2) and pG/7T (+5). After transient transfection, the expression level of reporter gene CAT in both pGBPCATdelta2 and its derivative plasmids was detected and analysed by CAT ELISA. RESULTS Identification by enzyme restrictions and PCR amplifications, as well as DNA sequencing confirmed that the plasmids were successfully constructed. Transfection of these plasmids and CAT detection suggested that insertion of 7cot into each point of GBP130 promoter enhanced the promoter activity, and downstream location of 7cot in the promoter showed higher promoter activity than those located upstream, with the strongest effect in Ins 4 (point +5). CONCLUSION Plasmid pG/7T (+5), in which 7cot was inserted into +5 point of GBP130 promoter, can be chosen as the responsive plasmid in establishing tetracycline-controlled transgenic expression system of malarial parasites.