PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNA(Leu) genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.