Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.