The smallest product of the Duchenne muscular dystrophy gene, dystrophin (Dp)71, is ubiquitously expressed in nonmuscle tissues. We previously showed that Dp71 expression in hepatic cells is modulated in part by stimulating factor 1 (Sp1), stimulating protein 3 (Sp3), and yin yang 1 (YY1) transcription factors, and that the polyaromatic hydrocarbon, β-naphthoflavone (β‑NF), downregulates Dp71 expression. The aim of the present study was to determine whether β‑NF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcription‑quantitative polymerase chain reaction was used to measure half‑life mRNA levels in β‑NF‑treated cells exposed to actinomycin D, an inhibitor of transcription, for 0, 4, 8, 12 and 16 h. Transient transfections with a plasmid carrying the Dp71 basal promoter fused to luciferase reporter gene were carried out in control and β‑NF‑treated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and β‑NF‑treated cells. To the best of our knowledge, the results demonstrated for the first time that this negative regulation takes place at the promoter level rather than the mRNA stability level. Interestingly, using EMSAs, β‑NF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that β‑NF may modulate the expression of other genes regulated by these transcription factors. In conclusion, β‑NF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.