In this study we demonstrated that ribonuclease A (RNase A) can recognize the thermodynamic asymmetry of siRNA duplexes, similar to other proteins involved in siRNA function such as argonaute 2. RNase A preferentially invades the siRNA duplex through the less stable terminus, i.e., the 3′ terminus of the sense strand. As evidence, only phosphorothioate (PS) modification at the sense strand overhang improved serum stability, whereas PS modification at the antisense strand overhang did not affect stability. Moreover, the improvement in stability caused by modification at the sense strand overhang was found to correlate with the terminal base pair composition of the siRNA. Gel electrophoresis and MALDI-TOF MS analysis indicated that modifications at the sense strand overhang improved the serum stability of siRNAs by inhibiting the directional invasion of RNase A. The blocking effect was not brought about by stabilization of the siRNA duplexes because there was no clear difference between the melting temperatures of siRNAs with PS modifications at each 3′ overhang.