Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.