Major vault protein (MVP) is the main constituent of the vault ribonucleoprotein particle and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several types of normal tissues, little is known about its physiological role. In the present study, we identified the crucial MVP promoter elements that regulate MVP expression. An examination of tissue expression profiles revealed that MVP was expressed in the heart, placenta, lung, liver, kidney and pancreas. Elements of the MVP promoter contain binding sites for transcription factors, STAT, p53, Sp1, E-box, GATA, MyoD and Y-box. By deletion analysis, a conserved proximal E-box binding site was demonstrated to be important for human MVP promoter transactivation. Introduction of siRNA against upstream stimulating factor (USF) 1, which is known to bind the E-box binding site, decreased the expression of MVP in SW620 and ACHN cells. Using a chromatin immunoprecipitation (ChIP) assay, USF1 bound the MVP promoter in SW620 cells. These findings suggest that USF1 binding to an E-box element may be critical for basal MVP promoter activation. The results of the present study are useful in understanding the molecular mechanisms regulating MVP gene expression, and may aid in elucidating the physiological functions of MVP.