Objective:To explore whether Cdk4 and CyclinD3 are involved in the inhibition of β-cell proliferation caused by miRNA-24. Methods:miRNA-24(miR-24)was highly expressed in MIN6 cells. Cell viability was determined using WST-1 assays. Cells were collected for flow cytometry analysis to test cell cycle distribution. Protein levels of Cdk4 and CyclinD3 were analyzed by Western blot. CyclinD3-pCMV5-myc and cdk4-pCMV5-myc recombinant plasmid were constructed and transfected into MIN6 cells together with pre-miR-24 or pre-Neg miRNA precursors. Cell proliferation was measured by the BrdU labeling. Results:MIN6 cells proliferation was reduced because of transfected miR-24,mainly due to G2 phase arrest. No changes of the level of apoptotic cells were detected. Cdk4 and CyclinD3 were downregulated by overexpressed miR-24. Overexpressed cdk4 and cyclinD3 reversed the β-cell proliferation inhibition. Conclusion:miR-24 inhibits β-cell proliferation by decreasing Cdk4 and CyclinD3 protein levels. The inhibition may be reversed by overexpressed cdk4 and cyclinD3.