AIM: To investigate the effect of cyclin B1 antisense oligodeoxynucleotide (ASON) with liposomal transfection on proliferation of HL60 cells. METHODS: After cyclin B1 ASON with liposomal transfection was used in vitro co-culture with HL60 cells,the protein and mRNA expression levels of cyclin B1 were measured by flow cytometry and RT-PCR. The cell apoptosis was detected by electron microscopy, in situ cell apoptosis detection kit (POD),DNA agarose gel electrophoresis and flow cytometry. RESULTS: In cyclin B1 ASON group the protein and mRNA expression levels of cyclin B1 were significantly inhibited than those in sense oligodeoxynucleotide (SON) group and blank group. Morever,when the ASON concentration increased,the proliferation ratio of HL60 cells and CFU-HL60 clony unit were also significantly inhibited. The apoptosis of HL60 cells was also observed. CONCLUSION: Cyclin B1 ASON specifically inhibited its protein and mRNA expression levels as well as the HL60 cell proliferations and induced leukemia cell apoptosis. It's effect depended upon the concentration of ASON.