Small interfering RNAs (siRNAs) are potentially powerful tools for therapeutic gene regulation. DNA cassettes encoding RNA polymerase III promoter-driven hairpin siRNAs allow long-term expression of siRNA in targeted cells. A variety of viral vectors have been used to deliver such cassettes to cells. Here we report on the development and use of a self-complementary recombinant adeno-associated virus (scAAV) vector for siRNA delivery into mammalian cells. We demonstrate that this modified vector efficiently delivers siRNA into multidrug-resistant human breast and oral cancer cells and suppresses MDR1 gene expression. This results in rapid, profound, and durable reduction in the expression of the P-glycoprotein multidrug transporter and a substantial reversion of the drug-resistant phenotype. This research suggests that scAAV-based vectors can be very effective agents for efficient delivery of therapeutic siRNA.