Transcription of transfer RNA (tRNA) requires sequential assembly of RNA polymerase III (Pol III) transcription factors ‐ TFIIIC and TFIIIB – followed by the recruitment of the Pol III enzyme complex. Recent studies have shown that in addition to producing tRNAs, Pol III transcription complexes assembled on DNA can exert “extra‐transcriptional” effects on chromosome function. Our study provides evidence of a unique extra‐transcriptional activity of assembled Pol III transcription complexes at a tRNA gene that blocks progression of intergenic RNA polymerase II transcription. We demonstrate that the Pol III transcription complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream non‐coding intergenic SUT467. Chromatin immunoprecipitation confirmed that the protection is predominately mediated by binding of the TFIIIB complex. Failure to block this readthrough results in compromised ATG31 translation. Since the Atg31p is required for autophagy in yeast, strains expressing the readthrough transcript exhibit reduced fitness under autophagy‐inducing conditions. Given the recent discovery of pervasive transcription in yeast, protection of neighboring genes from intergenic transcriptional interference may be a key extra‐transcriptional function of assembled Pol III complexes.