ABSTRACTThis study aimed to investigate the protective mechanisms of dexmedetomidine (Dex) and thrombopoietin (TPO) against hypoxia/reoxygenation (H/R)‐induced myocardial injury. Human cardiomyocyte AC16 cells were subjected to hypoxic conditions and treated with Dex and TPO. Cellular responses, including proliferation, apoptosis, and autophagy, were assessed. RNA sequencing and bioinformatic analyses were conducted to identify differentially expressed genes, followed by functional pathway enrichment analysis. The results demonstrated that Dex and TPO significantly promoted cell proliferation, reduced apoptosis and autophagy, and inhibited caspase‐3 activity and light chain 3B (LC3B) expression. Pathway enrichment analysis revealed the involvement of mitogen‐activated protein kinase (MAPK), transforming growth factor beta (TGF‐β), and tumor necrosis factor (TNF) signaling pathways. Although both treatments demonstrated overlapping effects, they also exhibited distinct gene regulation mechanisms. These findings suggested that Dex and TPO could mitigate H/R‐induced myocardial injury through complex gene regulatory mechanisms, highlighting their potential as therapeutic strategies for myocardial ischemia–reperfusion injury (MIRI).