This study identifies a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis that direct the cleavage of endogenous mRNAs. These siRNAs, derived from a noncoding RNA (At2g27400), differ from previously described regulatory small RNAs in two key respects: they require components of both the cosuppression pathway (RDR6 and SGS3) and the microRNA (miRNA) pathway (AGO1, DCL1, HEN1, and HYL1). These siRNAs repress the expression of genes that bear little overall resemblance to the genes from which they originate, a characteristic previously observed only for miRNAs. The identification of this silencing pathway provides new insights into posttranscriptional mRNA regulation in plants. The siRNAs are produced by Dicer-like enzymes from long double-stranded RNA precursors and obey the asymmetry rules that determine which strand enters the RISC complex. They target multiple members within a single gene family and require AGO1, DCL1, HEN1, and HYL1 for their biogenesis and function. Unlike miRNAs, these siRNAs derive from long double-stranded RNA and their accumulation depends on RDR6 and SGS3. The study also reveals that RDR6 and SGS3 are required for the accumulation of these siRNAs but have limited or no impact on the miRNA or chromatin siRNA pathways. The findings suggest that the AGO1-DCL1-HEN1-HYL1-SGS3-RDR6-dependent siRNA pathway is an ancient, conserved mechanism that regulates posttranscriptional gene silencing in plants.This study identifies a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis that direct the cleavage of endogenous mRNAs. These siRNAs, derived from a noncoding RNA (At2g27400), differ from previously described regulatory small RNAs in two key respects: they require components of both the cosuppression pathway (RDR6 and SGS3) and the microRNA (miRNA) pathway (AGO1, DCL1, HEN1, and HYL1). These siRNAs repress the expression of genes that bear little overall resemblance to the genes from which they originate, a characteristic previously observed only for miRNAs. The identification of this silencing pathway provides new insights into posttranscriptional mRNA regulation in plants. The siRNAs are produced by Dicer-like enzymes from long double-stranded RNA precursors and obey the asymmetry rules that determine which strand enters the RISC complex. They target multiple members within a single gene family and require AGO1, DCL1, HEN1, and HYL1 for their biogenesis and function. Unlike miRNAs, these siRNAs derive from long double-stranded RNA and their accumulation depends on RDR6 and SGS3. The study also reveals that RDR6 and SGS3 are required for the accumulation of these siRNAs but have limited or no impact on the miRNA or chromatin siRNA pathways. The findings suggest that the AGO1-DCL1-HEN1-HYL1-SGS3-RDR6-dependent siRNA pathway is an ancient, conserved mechanism that regulates posttranscriptional gene silencing in plants.