In this study, we introduced a pair of nucleotide enantiomers, D-/L-isonucleotides (D-/L-isoNA), to examine the interactions between siRNAs and their related proteins. The serum stability and gene-silencing activity of the modified siRNAs were systematically evaluated. Gene-silencing activity had a site-specific effect, and the incorporation of a single D-isoNA at the 8th position (counting from the 5′-terminus) in the antisense strand improved the gene-silencing activity by improving RISC loading and affecting the movement of the PIWI domain. D-isoNA incorporated at the terminus of siRNA including the 2nd position in the antisense strand and 3′-overhangs in the sense strand, especially the latter, enhanced nuclease resistance and prolonged the silencing retention time. In addition, L-isoNA incorporation into the middle of the sense strand enhanced activity. These results provide a chemical strategy for the modulation of siRNA gene-silencing activity and nuclease resistance.