Objective: To investigate the effect of down-regulation of HDAC6 expression on the biological behavior of breast cancer MCF-7 cells and the possible molecular mechanism. Methods: The breast cancer MCF-7 cells were transfected with HDAC6 siRNA or negative siRNA respectively, and the untreated MCF-7 cells were used as blank control. The gene and protein expressions of HDAC6 and p21 were determined by RT-PCR method and Western blot analysis, the cell proliferation and cell cycles were measured by CCK-8 assay and flow cytometry respectively, and the cell apoptosis was assessed by TUNEL staining. Results: The tests for validation of the interference demonstrated that mRNA and protein expression of HDAC6 were significantly decreased after HDAC6 siRNA transfection (both P<0.05), while the negative siRNA did not exert any effect (P>0.05). Compared with the blank control group, in HDAC6 siRNA group, the cell proliferation was significantly inhibited, the ratio of cells in the G0/G1 phase increased and in the S phase decreased, and the cell apoptosis rate significantly increased (all P<0.05), while the negative siRNA had none of the above effects (all P>0.05). Moreover, the gene and protein expression of p21 were significantly higher than those of the blank control group and negative siRNA group (both P<0.05). Conclusion: In breast cancer MCF-7 cells, down-regulation of HDAC6 expression can inhibit the cell proliferation, induce G0/G1 phase arrest and promote apoptosis, which may probably be associated with the elevation of p21 expression.