〔Abstract〕 Objective To investigate the function and mechanism of histone deacetylase 3(HDAC3) in modulating hypoxia-induced autophagy in H9 C2 cardiac cells. Methods The hypoxia model of H9 C2 was establishedin vitro. The expression and fluorescence activity of autophagy-related proteins LC3 B-Ⅱ, Beclin-1, and P62 were detected by Western blot and immunofluorescence staining. After the transfection of HDAC3 plasmid or interfering RNA, the expression and fluorescence activity of autophagy regulatory molecule mTOR and autophagy-related protein LC3 B were detected. Results Compared with the control group, the protein level of LC3 B-Ⅱ and Beclin-1 were augmented after hypoxia for 1 h, 6 h, and 12 h in H9 C2 cardiac cells(P<0.05), and the expression of p62 protein also increased first at 1 h and then decreased at 6 h(P<0.05). After hypoxia for 1 h and 6 h, the protein expression of HDAC3 and mTOR were amplified, while both of them declined at hypoxia for 12 h(P<0.05). The protein level and fluorescence activity of mTOR decreased, but the protein level and fluorescence activity of LC3 B increased after overexpression of HDAC3(P<0.05). Downregulation of HDAC3, the expression and fluorescence activity of mTOR enhanced, while the protein level and fluorescence activity of LC3 B reduced(P<0.05). Conclusion HDAC3 negatively regulates mTOR protein and promotes hypoxia-induced autophagy in H9 C2 cardiac cells.