RNA interference (RNAi) pathway is a gene silencing process during which small double-stranded RNA (dsRNA) molecules trigger the degradation of homologous RNA targets. Small interfering RNAs (siRNAs) are the mediators of the RNAi that can be induced in vitro and in vivo by direct application of chemically synthesized siRNAs. Recently, promoter-targeted small non-coding RNAs have been described to be capable of regulating gene expression at the transcriptional level. In the present study we tested the hypothesis that Dicer-substrate siRNAs (D-siRNAs), which trigger gene silencing through intrinsic RNAi pathway and are therefore more potent in gene knockdown than traditionally used 21-23mer siRNAs, may also display regulatory effects at the transcriptional level. Synthetic 27mer D-siRNAs targeting the promoter/early coding region of the CDKN2A gene were designed and transfected into HeLa cells, and 48 h post-transfection the CDKN2A expression was analyzed in quantitative real-time PCR using the housekeeping G6PDH gene as reference. In comparison to the mutant version, the CDKN2A gene was effectively knocked-down by the D-siRNA, but no evidence of transcriptional regulation was found. We conclude that the D-siRNA-induced suppression is likely to occur after transcription.