Short interfering RNAs (siRNAs) variously modified with 4′-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4′-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4′-thioribonucleosides. Since modifications of siRNAs with 4′-thioribonucleosides were well tolerated in RNAi activity compared with that of 2′-O-methyl nucleosides, 4′-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.