Most eukaryotic transcriptional regulators act in an RNA polymerase (Pol)-selective manner. Here we show that the human Maf1 protein negatively regulates transcription by all three nuclear Pols. Changes in Maf1 expression affect Pol I- and Pol III-dependent transcription in human glioblastoma lines. These effects are mediated, in part, through the ability of Maf1 to repress transcription of the TATA binding protein, TBP. Maf1 targets an Elk-1-binding site in the TBP promoter, and its occupancy of this region is reciprocal with that of Elk-1. Similarly, Maf1 occupancy of Pol III genes is inversely correlated with that of the initiation factor TFIIIB and Pol III. The phenotypic consequences of reducing Maf1 expression include changes in cell morphology and the accumulation of actin stress fibers, whereas Maf1 overexpression suppresses anchorage-independent growth. Together with the ability of Maf1 to reduce biosynthetic capacity, these findings support the idea that Maf1 regulates the transformation state of cells.