Stable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components. Display omitted •The impact of antisense transcription on protein levels is measured for 188 genes•Antisense has mostly weak suppressive effects on ∼25% of the genes•Regulation by antisense correlates with promoter overlap and H3K4 methylation•Antisense-regulated genes have lower than expected noise levels Huber et al. conduct a systematic study of the function of antisense RNAs in yeast by selectively suppressing the antisense transcripts of 188 GFP-tagged genes and quantifying the resulting changes in protein expression levels. The authors use this information to identify features that distinguish functional from non-functional antisense RNAs.