AbstractMost long non-coding RNAs (lncRNAs) encoded by eukaryotic genomes remain uncharacterized. Here we focus on a set of intergenic lncRNAs in fission yeast. Deleting one of these lncRNAs exhibited a clear phenotype: drug sensitivity. Detailed analyses of the affected locus revealed that transcription of the nc-tgp1 lncRNA regulates drug tolerance by repressing the adjacent phosphate-responsive permease gene transporter for glycerophosphodiester 1 (tgp1⁺). We demonstrate that the act of transcribing nc-tgp1 over the tgp1⁺ promoter increases nucleosome density, prevents transcription factor access and thus represses tgp1⁺ without the need for RNA interference or heterochromatin components. We therefore conclude that tgp1⁺ is regulated by transcriptional interference. Accordingly, decreased nc-tgp1 transcription permits tgp1⁺ expression upon phosphate starvation. Furthermore, nc-tgp1 loss induces tgp1⁺ even in repressive conditions. Notably, drug sensitivity results directly from tgp1⁺ expression in the absence of the nc-tgp1 RNA. Thus, transcription of an lncRNA governs drug tolerance in fission yeast.