RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of organisms. Recently, a small-interference RNA (siRNA) technology has been reported to produce post-transcriptional gene silencing in mammalian cells. In the present study, we constructed a human U6 promoter-driven mammalian expression vector to produce hairpin double-stranded RNA and transfected this into a human cell line. Using this siRNA system, we were able to knock down the gene expression of an enhanced green fluorescence protein. This result indicates that the plasmid vector-based siRNA system is a promising method to downregulate gene expression in human cells.