Recent genome-wide transcriptome studies suggest the presence of numerous bidirectional overlapping coding gene pairs in mammalian genomes. Various antisense RNAs are reported as non-coding RNAs that regulate the expression of sense RNA. However, it is still unclear whether the expression of bidirectional overlapping coding genes are regulated by the opposite strand gene transcript acting as a non-coding RNA. Bop1 and Scx are a pair of bidirectional overlapping coding genes related to cellular proliferation and differentiation, respectively. Scx gene is localized in the intron 3 region of the Bop1 gene. The expression of these genes is reciprocally regulated by estrogen (E2) in the mouse uterus. In situ hybridization indicated that both genes are expressed in the uterine endometrial epithelial cells and that the antisense RNA of Scx (Bop1 intronic RNA) accumulates as a stable RNA in these cells. The existence of Bop1 intronic RNA was confirmed by reverse transcription-PCR and was increased after E2 treatment, coinciding with a decrease in Scx mRNA. Murine myoblasts expressing doxycycline-inducible endogenous Bop1 gene showed an increase in Bop1 intronic RNA and a simultaneous decrease in Scx mRNA. Murine fibroblasts expressing Scx mRNA from an exogenous Scx mini-gene indicated that the accumulation of Bop1 intronic RNA impairs the Scx gene expression in a trans-acting manner, which resulted in the reduction of the Scx mRNA level. This study demonstrates a novel example of hormone-stimulated intronic non-coding RNA down-regulating the expression of an opposing strand-overlapping coding gene.