Murine T-bet (T-box expressed in T cells) is a master regulator of IFN-γ gene expression in NK and T cells. T-bet also plays a critical role in autoimmunity, asthma and other diseases. However, cis elements or trans factors responsible for regulating T-bet expression remain largely unknown. Here, we report on our discovery of six Sp1-binding sites within the proximal human T-BET promoter that are highly conserved among mammalian species. Electrophoretic mobility shift assays demonstrate a physical association between Sp1 and the proximal T-BET promoter with a direct dose response between Sp1 expression and T-BET promoter activity. Ectopic overexpression of Sp1 also enhanced T-BET expression and cytokine-induced IFN-γ secretion in NK cells and T cells. Mithramycin A, which blocks the binding of Sp1 to the T-BET promoter, diminished both T-BET expression and IFN-γ protein production in monokine-stimulated primary human NK cells. Collectively, our results suggest that Sp1 is a positive transcriptional regulator of T-BET. As T-BET and IFN-γ are critically important in inflammation, infection, and cancer, targeting Sp1, possibly with mithramycin A, may be useful for preventing and/or treating diseases associated with aberrant T-BET or IFN-γ expression.