Development of RNA interference as a novel class of therapeutics requires improved pharmacokinetic properties of short interfering RNA (siRNA). To confer enhanced serum stability to Sur10058, a hyperfunctional siRNA which targets survivin mRNA, a systematic modification at the 2′-sugar position and phosphodiester linkage was introduced into Sur10058. End modification of three terminal nucleotides by 2′-OMe and phosphorothioate substitutions resulted in a modest increase in serum stability, with 3′ end modification being more effective. Alternating modification by 2′-OMe substitution significantly stabilized Sur10058, whereas phosphorothioate modification was only marginally effective. Through various combinations of 2′-OMe, 2′-F and phosphorothioate modifications that were directed mainly at pyrimidine nucleotides, we have identified several remarkably stable as well as efficient forms of Sur10058. Thus, our results provide an effective means to stabilize siRNA in human serum without compromising the knockdown efficiency. This advancement will prove useful for augmenting the in vivo potency of RNA interference.