Abstract: Objective To investigate the specificity and regulatory element of human mammaglobin A (MGA) promoter, and validate its expression activity in living breast tumor tissues. Methods The 5.6kb MGA promoter was amplified by PCR from human genome. Then, four recombinant plasmids with different fragment or element mutation of MGA promoter driving firefly luciferase reporter were constructed. After transient transfection into breast cancer cell line T47D and human embryonic kidney cell line HEK293T, dual luciferase reporter gene system was used to determine the activity of these MGA promoter fragments. T47D cells stably transfected with MGA recombinant vector(T47DEM) was implanted subcutaneously in nude mice, and the subcutaneous breast tumor tissue were imaged by in vivo imaging system(IVIS). Results (1) Superposition of two enhancers markedly reduced the promoter activity by 82%; (2) Site-specific deletion of YY1 binding element decreased both activity (76.2%) and specificity (38.4%) of MGA promoter; (3) The deletion of Nkx2.5 binding site had no effect on the activity of MGA promoter but significantly reduced the specificity (31.9%); (4) In vivo image of the subcutaneous breast tumor tissue developed form (T47DEM) showed obviously the firefly luciferase report gene activity. Conclusion The effect of two transcription factor binding sites(YY1 and Nkx2.5) and superimposing two enhancers to MGA promoter are investigated and the constructed T47DEM cell line could be used for the in vivo drug screening of breast cancer.