Groucho (Gro) is a Drosophila melanogaster transcriptional corepressor that directly interacts with the histone deacetylase Rpd3. Although previous studies suggest that this interaction is required for repression of Gro-responsive reporters in cultured cells, the in vivo significance of this interaction and the mechanism by which it leads to repression remain largely unexplored. In this study, we show that Gro is partially dependent on Rpd3 for repression, supporting the idea that Rpd3-mediated repression is one mode of Gro-mediated repression. We demonstrate that Gro colocalizes with Rpd3 to the chromatin of a target gene and that this is accompanied by the deacetylation of specific lysines within the N-terminal tails of histones H3 and H4. Gro overexpression leads to wing patterning defects and ectopic repression in the wing disc of transcription directed by the vestigial quadrant enhancer. These effects are reversed by the histone deacetylase inhibitors TSA and HC-Toxin and by the reduction of Rpd3 gene dosage. Furthermore, repression of the vestigial quadrant enhancer is accompanied by a Gro-mediated increase in nucleosome density, an effect that is reversed by histone deacetylase inhibitors. We propose a model in which Gro-mediated histone deacetylation results in increased nucleosome density leading to transcriptional repression.