[Purpose] To investigate the silencing effect of cyclin D1 gene in leukemia K562 cells by RNA interference technique and the influeuce on cell growth, cell cycle and apoptosis. [Methods] Plasmid vectors expressing small hairpin RNA (shRNA) targeting at cyclin D1 gene were constructed and transfected into K562 cells mediated by chitosan. Expression of Cyclin D1 protein before and after transfection was examined using Western blot analysis. Cellular proliferation was evaluated by soft agar colony formation assay. The cell cycle and apoptosis were determined by flow cytometry. [Results] Expression of cyclin D1 protein was markedly down-regulated, proliferation of K562 cells was inhibited, and capability of colony formation was suppressed after transfection with pshRNA-419 and pshRNA-575 mediated by chitosan. K562 cells were arrested at G0/G1 phase, and cell apoptosis was induced significantly. But no above biological effects was found in m-pshRNA-790 plasmid. [Conclusion] Down-regulation of cyclin D1 may inhibit growth of K562 cells, and induce cell apoptosis. It suggests that cyclin D1 gene might serve as an effective target for the treatment of leukemia.