RNA interference (RNAi) is a well-established research tool and is also maturing as a novel therapeutic approach. For the latter, microRNA-like off-target activity of short interfering RNAs (siRNAs) remains as one of the main problems limiting RNAi drug development. In this communication, we report that replacement of a single internucleoside phosphodiester in the seed region (nucleotides 2 to 7) of the guide strand with an amide linkage suppressed the undesired microRNA-like off-target activity by at least an order of magnitude. For the specific siRNA targeting the PIK3CB gene, an amide modification between the third and fourth nucleotides of the guide strand showed the strongest enhancement of specificity (completely eliminated off-target silencing) while maintaining high on-target activity. These results are important because off-target activity is one of the main remaining roadblocks for RNA based drug development.