Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. This study identifies a new class of endogenous small interfering RNAs (siRNAs) in Arabidopsis that direct the cleavage of endogenous mRNAs. These siRNAs are derived from a noncoding RNA (At2g27400) and are processed into 21-nucleotide siRNAs. They differ from previously described regulatory small RNAs in that they require components of the cosuppression pathway (RDR6 and SGS3) and the miRNA pathway (AGO1, DCL1, HEN1, and HYL1), but not components needed for heterochromatic siRNAs (DCL3 and RDR2). Additionally, these siRNAs repress genes that are not similar to the genes from which they originate, a characteristic previously observed only for miRNAs. The identification of this silencing pathway provides a new dimension to posttranscriptional mRNA regulation in plants. The siRNAs are produced through a pathway that requires AGO1, DCL1, HEN1, HYL1, SGS3, and RDR6. This pathway is similar to the miRNA pathway but differs from the heterochromatic siRNA pathway. These siRNAs target complementary mRNAs and regulate their expression, suggesting that numerous endogenous genes may be regulated by a similar pathway. The study also shows that these siRNAs are involved in the regulation of gene expression in plants, and that their accumulation is affected by mutations in specific genes. The findings highlight the importance of these siRNAs in plant development and gene regulation.Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. This study identifies a new class of endogenous small interfering RNAs (siRNAs) in Arabidopsis that direct the cleavage of endogenous mRNAs. These siRNAs are derived from a noncoding RNA (At2g27400) and are processed into 21-nucleotide siRNAs. They differ from previously described regulatory small RNAs in that they require components of the cosuppression pathway (RDR6 and SGS3) and the miRNA pathway (AGO1, DCL1, HEN1, and HYL1), but not components needed for heterochromatic siRNAs (DCL3 and RDR2). Additionally, these siRNAs repress genes that are not similar to the genes from which they originate, a characteristic previously observed only for miRNAs. The identification of this silencing pathway provides a new dimension to posttranscriptional mRNA regulation in plants. The siRNAs are produced through a pathway that requires AGO1, DCL1, HEN1, HYL1, SGS3, and RDR6. This pathway is similar to the miRNA pathway but differs from the heterochromatic siRNA pathway. These siRNAs target complementary mRNAs and regulate their expression, suggesting that numerous endogenous genes may be regulated by a similar pathway. The study also shows that these siRNAs are involved in the regulation of gene expression in plants, and that their accumulation is affected by mutations in specific genes. The findings highlight the importance of these siRNAs in plant development and gene regulation.