Abstract: Objective To investigate the relationship between the expression of glucosylceramide synthase (GCS) mRNA in vincristine-resistant KBV₂₀₀ human cancer cell line and multidrug resistance (MDR) of the cancer cells. Methods Reverse transcriptional polymerase chain reaction (RT-PCR) was employed to analyze the differential expression of GCS mRNA between KBV₂₀₀ and KB cell lines and the changes in the mRNA expressions of GCS and mdrl gene in KBV₂₀₀ cells after reversion of MDR. The effects of de-phenyl-z-palmaitoylamino-3-morpholine-1-propanol (DL-PPMP) and verapamil in reversing MDR of the cells were evaluated by MTT assay. Results KBV₂₀₀ cells exhibited significantly increased expressions of GCS and mdrl gene, whereas mdrl gene failed to be detected in the parental KB cells. DL-PPMP within the concentrations ranging from 5 to 25 μmol/L could inhibit the expression of GCS gene, with the maximum inhibition achieved at 25 μmol/L. Verapamil at the concentration of 10 μmol/L was already sufficient to induce inhibition of GCS expression in KVB₂₀₀ cells, which was more manifest with the concentration of 15 μmol/L. DL-PPMP and verapamil were found to inhibit mdrl gene expression in KBV₂₀₀ cells at the mRNA level, and complete inhibition occurred after a 48-hour DL-PPMP treatment at 25 μmol/L. Conclusion The inhibition of GCS and mdrl gene expressions is positively correlated with the concentrations of DL-PPMP and verapamil, which can reverse MDR by inhibiting synthesis of GCS and mdrl gene, indicating the positive correlation between the expression of GCS gene and MDR in KBV₂₀₀ cells. GCS gene might play an importantrole in MDR during tumor progression.