Background: Resistance to chemotherapeutic agents is one of the major obstacles in the treatment of laryngeal cancer. Upregulated expression of P-glycoprotein (Pgp), the MDR1 gene product, confers multidrug resistance (MDR) to laryngeal cancer cells. In this study, we reversed the resistance to chemotherapeutic agents in human laryngeal cancer cells (LSC-1/TAX) by using the lentivirus-based small hairpin interference ribonucleic acid (shRNA) technique. Methods: LSC-1/TAX cells were transfected with lentivirus vector that contains the shRNA construct targeting MDR1 messenger ribonucleic acid (mRNA). The knockdown of MDR1 gene expression was assessed by reverse transcription polymerase chain reaction (RT-PCR), and the protein level of Pgp was determined by Western blot and immunocytochemistry. Sensitization of the laryngeal cancer cells to various antineoplasm agents will be quantified by methylthiazolyl tetrazolium (MTT) assays. Apoptosis was analyzed by flow cytometry. Results: It was shown that MDR1 shRNA transfection reduced the MDR1 gene expression in LSC-1/TAX cells by RT-PCR experiment. Western blot and immunocytochemistry showed that Pgp expression was significantly and specifically inhibited. MTT assay revealed a reverse of MDR1 gene-dependent MDR in the laryngeal cancer cells using shRNA lentivirus vectors. Increased apoptosis has been observed in the MDR1 knockdown LSC-1/TAX cells when exposed to paclitaxel (TAX) treatment. Conclusions: MDR1 shRNA lentivirus vectors can significantly inhibit MDR1 expression at both mRNA and protein levels. Inhibition of MDR1 gene expression conferred an increased sensitivity to drug resistance in laryngeal cancer cells to conventional chemotherapeutic agents.