Natural antisense transcripts (NATs) are RNA molecules that originate from opposite DNA strands of the same genomic locus (cis-NAT) or unlinked genomic loci (trans-NAT). NATs may play various regulatory functions at the transcriptional level via transcriptional interference. NATs may also regulate gene expression levels post-transcriptionally via induction of epigenetic changes or double-stranded RNA formation, which may lead to endogenous RNA interference, RNA editing or RNA masking. The true biological significance of the natural antisense transcripts remains controversial despite many years of research. Here, we summarize the current state of knowledge and discuss the sense-antisense overlap regulatory mechanisms and their potential.