RNA silencing is a eukaryotic defense mechanism that processes double-stranded RNA (dsRNA) into short interfering RNA (siRNA) of 21–26 nt. These siRNAs target and degrade genes matching the dsRNA. In plants, additional silencing processes involve systemic spread and RNA-directed DNA methylation. This study shows that siRNAs from a green fluorescent protein (GFP) transgene exist in two size classes: short (21–22 nt) and long (24–26 nt), while those from endogenous retroelements are only long. Viral suppressors and Arabidopsis mutations indicate these classes have different roles. Long siRNA is not essential for mRNA degradation but correlates with systemic silencing and DNA methylation. Short siRNA is involved in mRNA degradation but not systemic signaling or methylation. These findings reveal complexity in plant RNA silencing, possibly applicable to animals. The study also shows that retroelements produce only long siRNA, and that long siRNA is associated with DNA methylation. The results suggest that short siRNA is incorporated into RISC for mRNA degradation, while long siRNA plays a role in systemic signaling and DNA methylation. The study highlights the importance of siRNA size classes in RNA silencing mechanisms and their roles in plant defense against viruses and transposable elements.RNA silencing is a eukaryotic defense mechanism that processes double-stranded RNA (dsRNA) into short interfering RNA (siRNA) of 21–26 nt. These siRNAs target and degrade genes matching the dsRNA. In plants, additional silencing processes involve systemic spread and RNA-directed DNA methylation. This study shows that siRNAs from a green fluorescent protein (GFP) transgene exist in two size classes: short (21–22 nt) and long (24–26 nt), while those from endogenous retroelements are only long. Viral suppressors and Arabidopsis mutations indicate these classes have different roles. Long siRNA is not essential for mRNA degradation but correlates with systemic silencing and DNA methylation. Short siRNA is involved in mRNA degradation but not systemic signaling or methylation. These findings reveal complexity in plant RNA silencing, possibly applicable to animals. The study also shows that retroelements produce only long siRNA, and that long siRNA is associated with DNA methylation. The results suggest that short siRNA is incorporated into RISC for mRNA degradation, while long siRNA plays a role in systemic signaling and DNA methylation. The study highlights the importance of siRNA size classes in RNA silencing mechanisms and their roles in plant defense against viruses and transposable elements.