RNA silencing is a eukaryotic genome defense mechanism that involves the processing of double-stranded RNA (dsRNA) into 21–26 nt short interfering RNA (siRNA). The siRNA mediates gene silencing by targeting and degrading complementary mRNAs. In plants, additional silencing processes include systemic spread of silencing and RNA-directed DNA methylation. This study demonstrates that siRNAs produced from a green fluorescent protein (GFP) transgene in plants are divided into two size classes: short (21–22 nt) and long (24–26 nt). In contrast, siRNAs from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these siRNA classes have distinct roles. The long siRNA is essential for systemic silencing and DNA methylation, while the short siRNA is involved in mRNA degradation. These findings reveal a complex regulatory network in plant RNA silencing, which may also apply to animals.RNA silencing is a eukaryotic genome defense mechanism that involves the processing of double-stranded RNA (dsRNA) into 21–26 nt short interfering RNA (siRNA). The siRNA mediates gene silencing by targeting and degrading complementary mRNAs. In plants, additional silencing processes include systemic spread of silencing and RNA-directed DNA methylation. This study demonstrates that siRNAs produced from a green fluorescent protein (GFP) transgene in plants are divided into two size classes: short (21–22 nt) and long (24–26 nt). In contrast, siRNAs from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these siRNA classes have distinct roles. The long siRNA is essential for systemic silencing and DNA methylation, while the short siRNA is involved in mRNA degradation. These findings reveal a complex regulatory network in plant RNA silencing, which may also apply to animals.