Atherosclerotic cardiovascular disease and acute myocardial infarction are the leading causes of mortality worldwide, and apoptosis is the major pathway of cardiomyocyte death under hypoxic conditions. Although studies have reported changes in the expression of certain pro‑apoptotic and anti‑apoptotic genes in hypoxic cardiomyocytes, genetic regulations are complex in human cardiomyocytes and there is much that remains to be fully elucidated. The present study aimed to identify differentially expressed genes in hypoxic human AC16 cardiomyocytes using next‑generation sequencing and bioinformatics. A total of 24 genes (15 upregulated and 9 downregulated) with potential micro (mi)RNA‑mRNA interactions were identified in the miRmap database. Utilising the Gene Expression Omnibus database of cardiac microvascular endothelial cells, tensin 1, B‑cell lymphoma 2‑interacting protein 3 like, and stanniocalcin 1 were found to be upregulated, and transferrin receptor and methyltransferase like 7A were found to be downregulated in response to hypoxia. Considering the results from miRmap, TargetScan and miRDB together, two potential miRNA‑mRNA interactions were identified: hsa‑miRNA (miR)‑129‑5p/CDC42EP3 and hsa‑miR‑330‑3p/HELZ. These findings contribute important insights into possible novel diagnostic or therapeutic strategies for targeting cardiomyocytes under acute hypoxic stress in conditions, including acute myocardial infarction. The results of the present study also introduce an important novel approach in investigating acute hypoxic pathophysiology.