Multidrug resistance (MDR) is a serious obstacle to cancer chemotherapy. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers MDR to tumor cells. This study explored the possibility of reducing drug resistance by targeting the mdr1 gene using short hairpin RNA (shRNA). Two different shRNAs were designed and constructed in a pSilencer 2.1-U6 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. Real-time polymerase chain reaction and Western blotting were used to characterize the inhibited expression of MDR1 mRNA and P-gp, and the drug sensitivity of the transfected cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the inhibition of P-gp expression by small interfering RNA selectively restored sensitivity to the drugs transported by P-gp. Evaluation of chemosensitivity showed 52.58% reversal by p2.1-shRNA1 and 73.07% reversal by p2.1-shRNA2 in drug resistance for harringtonine, and 84.87% reversal by p2.1-shRNA1 and 94.23% reversal by p2.1-shRNA2 in drug resistance for curcumin in the transfected cells. The results demonstrated the efficacy and selectivity of shRNA in reversing MDR in drug-resistant HT9 cells in vitro.