Specific knock-down of cellular gene expression using small interfering RNAs (siRNAs) is a powerful gene silencing technique in mammalian cells. Early siRNAs were double stranded, and 19-21bp in length, but several variations in siRNA structure have been introduced to achieve better silencing efficiency. In addition, siRNA modules have been incorporated into higher-order RNA structures to generate multi-functional RNA molecules. The effects of such structural variations on the activities of siRNAs have not been thoroughly studied. Here, we present a structure-activity relationship study of siRNA structural variants. Specifically, we focus on the effect on silencing efficiency of the attachment of extra, target-unrelated sequences to the conventional short duplex siRNA structure. Interestingly, while some siRNA structural variants efficiently silence target gene expression, others show a reduction in or a complete lack of silencing activity. Off-target effects and innate immune responses triggered by siRNA structural variants were also measured. In vitro Dicer cleavage reactions show that all siRNA structural variants are substrates of Dicer, but digestion patterns vary. To our knowledge, this is the first systematic structure-activity relationship analysis of siRNAs bearing structural variations. Our results provide useful guidelines for the design of siRNA structural variants and for the construction of complex RNA molecules bearing functional siRNA modules.