This study was purposed to explore the expression of glucosylceramide synthase (GCS) in human leukemia cells and its relationship with multidrug resistance. RT-PCR was used to analyze peripheral blood samples from 53 leukemia patients with multidrug resistance/non-resistance, and to detect the expression level of GCS gene in HL-60 cells and HL-60/ADR cells, the expression level was compared with the level of mdr-1. The expressions of GCS protein and P-gp protein in HL-60 cells and HL-60/ADR cells were assayed by Western blot analysis. The results showed that the relative optical density ratio of GCS gene amplified bands in samples of leukemia patients with drug-resistance was significantly higher than that in samples of leukemia patients with drug non-resistance group (P < 0.05), meanwhile the significant enhancement of optical density value of GCS gene amplified bands accompanied by high expression of mdr-1 gene. Their correlation showed positive (P < 0.01, r = 0.6). The GCS mRNA and protein were overexpressed in HL-60/ADR cells, and their expression levels were obviously higher than that in HL-60 cells, meanwhile the expression of mdr-1 mRNA and P-gp also significantly increased in HL-60/ADR cells. It is concluded that the high level of GCS in leukemia patients possibly is associated with multidrug resistance of leukemia cells.