MicroRNAs (miRNAs) are aberrantly expressed in the pathophysiologic process of heart failure (HF). However, the functions of a certain miRNA in different cardiac cell types during HF are scarcely reported, which might be covered by the globe effects of it on the heart. In the current study, Langendorff system was applied to isolate cardiomyocytes (CMs) and cardiac fibroblasts (CFs) from transverse aortic constriction (TAC)-induced mice. Slight increase of miR-320 expression was observed in the whole heart tissue of TAC mice. Interestingly, miR-320 was significantly elevated in CMs but decreased in CFs from TAC mice at different time points. Then, recombinant adeno-associated virus 9 with cell-type-specific promoters were used to manipulate miR-320 expressions in vivo. Both in vitro and in vivo experiments showed the miR-320 overexpression in CMs exacerbated cardiac dysfunction, whereas overexpression of miR-320 in CFs alleviated cardiac fibrosis and hypertrophy. Mechanically, downstream signaling pathway analyses revealed that miR-320 might induce various effects via targeting PLEKHM3 and IFITM1 in CMs and CFs, respectively. Moreover, miR-320 mediated effects could be abolished by PLEKHM3 re-expression in CMs or IFITM1 re-expression in CFs. Interestingly, miR-320 treated CFs were able to indirectly affect CMs function, but not vice versa. Meanwhile, upstream signaling pathway analyses showed that miR-320 expression and decay rate were rigorously manipulated by Ago2, which was regulated by a cluster of cell-type-specific TFs distinctively expressed in CMs and CFs, respectively. Together, we demonstrated that miR-320 functioned differently in various cell types of the heart during the progression of HF.