Myocardial infarction (MI) is an important cause of cardiovascular disease. microRNAs (miRNAs) have been indicated as pivotal regulators in the physiological and pathological processes of heart diseases. The purpose of this study was to investigate the role of miR-140-5p in hypoxia-induced cell injury in H9c2 cells and its underlying mechanism. H9c2 cells were subjected to hypoxia, before which the expression levels of miR-140-5p and MLK3 were overexpressed or knocked down through transient transfection. The efficiency of transfection was verified by qRT-PCR and Western blotting. Cell viability, apoptotic cell rate, and the expression changes of apoptosis-related proteins were determined by trypan blue exclusion, flow cytometry, and Western blotting, respectively. Furthermore, Western blotting was performed to assess the expression levels of core factors related with p38MAPK and JNK signaling pathways. As a result, hypoxia significantly reduced cell viability and increased cell apoptosis in H9c2 cells. miR-140-5p inhibition attenuated cell injury induced by hypoxia in H9c2 cells, while miR-140-5p overexpression expedited the cell injury, as evidenced by the decreased cell viability and enhanced cell apoptosis. Moreover, miR-140-5p promoted the activation of p38MAPK and JNK pathways. miR-140-5p positively modulated the expression of MLK3. ML3 overexpression reversed the regulatory effects of miR-140-5p inhibition on hypoxia-injured H9c2 cells. In conclusion, this study demonstrated that miR-140-5p aggravated hypoxia-induced cell injury partially through up-regulation of MLK3.