Ischemic heart disease, especially myocardial infarction, poses a serious risk to human health. S100 calcium-binding protein A12 (S100A12) expression was previously reported to be upregulated in ST-segment elevation myocardial infarction. Therefore, the present study investigated the role of S100A12 in hypoxia/reoxygenation (H/R)-induced cardiomyocytes injury and the associated mechanism. An in vitro H/R-induced cardiomyocyte injury model was first established using AC16 cells. The expression level was found to be hugely upregulated in H/R-induced AC16 cells. Subsequently, cell transfection was conducted to knock down the expression level of S100A12, and the following cellular biological assays revealed that S100A12 knockdown could not only inhibit H/R-induced AC16 cell injury by improving cell viability and decreasing the release of lactate dehydrogenase, as well as reducing apoptotic cells, but also reduce the production of inflammatory cytokines (TNF-α, IL-1β and IL-6), restore the balance of oxidation-antioxidant factors (malondialdehyde, superoxide dismutase and glutathione), and inhibit the activated pyroptosis upon H/R induction. Then, co-immunoprecipitation was used to verify the interaction between S100A12 and caspase-4 (CASP4). CASP4 overexpression reversed the inhibitory effects of S100A12 downregulation on H/R-induced cardiomyocyte injury. In conclusion, these results suggest that S100A12 knockdown can ameliorate H/R-induced cardiomyocyte injury by regulating CASP4 expression. Therefore, S100A12 serves as a potential therapeutic target for the treatment of myocardial ischemia/reperfusion injury.