The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5′ phosphate to achieve activity in vivo, and we developed a metabolically stable 5′-(E)-vinylphosphonate (5′-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism. Display omitted ► Chemically modified single-stranded siRNAs silence gene expression in animals ► ss-siRNAs require 5′ phosphate and association with AGO2 for gene silencing ► Passenger strand is dispensable for potent RNAi-mediated gene silencing ► Single-stranded siRNAs provide an alternate option for RNAi therapeutics Chemically modified RNA oligonucleotides load into Ago2-containing RISC complexes to effect RNA silencing without the need for a passenger strand. These single-stranded siRNAs can be applied directly in animals and thus offer new alternatives for developing therapeutics and diagnostics.