OBJECTIVE To investigate the effect of antisense cDNA of cyclin D1 on the cyclin D1 gene expression and cell proliferation of human hepatocarcinoma HepG2 cells in vitro. METHODS Plasmids containing cyclin D1 antisense cDNA were constructed and transfected into HepG2 cells. Their effects on cell proliferation were examined by MTT method, RT-PCR, immunohistochemical means, and flow cytometry. RESULTS Cyclin D1 antisense cDNA significantly inhibited the growth of HepG2 cells. The inhibition peaked at 48 hour after transfection by MTT method. RT-PCR analysis showed that cyclin D1 antisense cDNA down-regulated cyclin D1 at the mRNA levels. Expression level of cyclin D1 protein was also decreased as shown by immunohistochemical studies. Cell-cycle analysis by flow cytometry showed that transfected HepG2 cells were arrested at the G1 phase of the cell cycle. CONCLUSIONS Our data suggest that cyclin D1 antisense cDNA could specifically inhibit the expression of cyclin D1 mRNA and protein and regulate cell cycle and cell proliferation of HepG2 cells. Cyclin D1 antisense cDNA may serve as a potential antitumor strategy in regulating cell-cyclin treating advanced HCCs.