Abstract For a significant fraction of mRNAs, their expression is regulated by other RNAs, including cis natural antisense transcripts (cis-NATs) that are complementary mRNAs transcribed from opposite strands of DNA at the same genomic locus. The regulatory mechanism of mRNA expression by cis-NATs is unknown, although a few possible explanations have been proposed. To understand this regulatory mechanism, we conducted a large-scale analysis of the currently available data and examined how the overlapping arrangements of cis-NATs affect their expression level. Here, we show that for both human and mouse the expression level of cis-NATs decreases as the length of the overlapping region increases. In particular, the proportions of the highly expressed cis-NATs in all cis-NATs examined were ∼36 and 47% for human and mouse, respectively, when the overlapping region was <200 bp. However, both proportions decreased to virtually zero when the overlapping regions were >2000 bp in length. Moreover, the distribution of the expression level of cis-NATs changes according to different types of the overlapping pattern of cis-NATs in the genome. These results are consistent with the transcriptional collision model for the regulatory mechanism of gene expression by cis-NATs.