Non-coding (nc)RNAs are key players in numerous biological processes such as gene regulation, chromatin domain formation and genome stability(1,2). Large ncRNAs interact with histone modifiers' and are involved in cancer development(6), X-chromosome inactivation(7) and autosomal gene imprinting(8). However, despite recent evidence showing that pervasive transcription is more widespread than previously thought(9), only a few examples mediating gene regulation in eukaryotes have been described(10). In Saccharomyces cerevisiae, the bona-fide regulatory ncRNAs are destabilized by the Xrn1 5'-3' RNA exonudease(11,12) (also known as Kem1), but the genome-wide characterization of the entire regulatory ncRNA family remains elusive. Here, using strand-specific RNA sequencing (RNA-seq), we identify a novel class of 1,658 Xrn1-sensitive unstable transcripts (XUTs) in which 66% are antisense to open reading frames. These transcripts are polyadenylated and RNA polymerase II (RNAPII)-dependent. The majority of XUTs strongly accumulate in lithium-containing media, indicating that they might have a role in adaptive responses to changes in growth conditions. Notably, RNAP II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis of Xrn1-deficient strains revealed a significant decrease of RNAPII occupancy over 273 genes with antisense XUTs. These genes show an unusual bias for H3K4me3 marks and require the Set1 histone H3 lysine 4 methyl-transferase for silencing. Furthermore, abolishing H3K4me3 triggers the silencing of other genes with antisense XUTs, supporting a model in which H3K4me3 antagonizes antisense ncRNA repressive activity. Our results demonstrate that antisense ncRNA-mediated regulation is a general regulatory pathway for gene expression in S. cerevisiae.