We have identified an RNA silencing inducible sequence (RSIS) in rice, and developed a new RNA silencing system which is induced by simply linking RSIS to a target gene sequence. Two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5’ or 3’ end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. This system does not require the formation of inverted repeat or hairpin structures, and is not associated with methylation of the target gene promoter. These transgenic plants accumulated 21-24 nt small RNAs from target genes and from transgenes containing RSIS. RSIS-mediated RNA silencing did not affect expression level of target pre-mRNA (unspliced form), but decreased target mRNA (spliced form), in isolated nuclei. RSIS-mediated RNA silencing is nuclear post-transcriptional gene silencing. Small RNA sequencing revealed that the region generating siRNAs extended to 3’ distal region of transgene terminator. Correspondingly, transcriptional read-through occurred in RSIS containing transgenes. These results suggest that RSIS prevents transciption termination at proper site of RSIS-containing transgene transcript, and aberrant transgene transcripts without poly (A) tail are lead to produce primary siRNAs, resulting in target degradation via PTGS.