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DsRNAs longer than 30 bp induce interferon response and global changes in gene expression profile in mammalians. 21 bp siRNA and 25/27 bp dsiRNA acting via RNA interference mechanism are used for specific gene silencing in this class of organisms. We designed selectively 2′-O-methyl-modified 42 and 63 bp anti-MDR1-siRNAs that silence the expression of P-glycoprotein and restore the sensitivity of drug-resistant cancer cells to cytostatic more efficiently than canonical 21 bp siRNAs. We also show that they act in a Dicer-independent mode and are devoid of immunostimulating properties. Our findings suggest that 42 and 63 bp siRNAs could be used as potential therapeutics.