Non-secreted macrophage colony-stimulating factor(M-CSF) plays an important role in genesis and progression of tumors. To explore the regulation of cytoplasmic M-CSF on the proliferation of HeLa cells, pCMV/myc/cyto-M-CSF vectors were transfected into HeLa cells. After comfirmed by RT-PCR, Western blot and immunocytochemistry, the effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by MTT and antisense oligonucleotides. The doubling time was counted according to cell growth curves. The mRNA expression of cyclinE, cyclinD1/2/3, CDK2/4/6 were assayed by RT-PCR. The results from RT-PCR, Western blot and immunocytochemistry showed that both M-CSF mRNA and protein were expressed at a higher level and localized to the cytoplasm in M-CSF-transfected HeLa cells, compared with either pCMV/myc/cyto-transfected HeLa cells or untransfected HeLa cells. M-CSF-transfected HeLa cells had shorter doubling time and more significantly augmented reproductive activity than either pCMV/myc/cyto-transfected HeLa cells or untransfected HeLa cells. M-CSF specific antisense oligonucleotides significantly inhibited the proliferation of the M-CSF-transfected cells, but had little effect on the other two groups. Furthermore, cytoplasmic M-CSF up-regulated the mRNA expression of cyclinD1, cyclinD3, CDK2 and CDK6(P < 0.05). So it was concluded that cytoplasmic M-CSF accelerates the proliferation of HeLa cells by up-regulating the mRNA expression of cyclinD1/D3 and CDK2/6.