Background <p>Pathological myocardial hypertrophy is a key risk factor for heart failure and cardiovascular death. MicroRNAs (miRNAs), as a key post transcriptional regulator of gene expression, play an increasingly important role in myocardial hypertrophy and injury. This study aimed to investigate the expression, function, and mechanism of miR-32-3p through the miR-32-3p/ATF4 axis in ISO induced injury of human AC16 cardiomyocytes.</p> Methods <p>A cell injury model was established by treating AC16 cells with 1 µM ISO. miR-32-3p mimic/inhibitor were transfected. Gene expression levels were detected using qRT-PCR; cell proliferation was evaluated via the CCK-8 assay; the levels of inflammatory cytokines (IL-6, TNF-α, IL-1β) were detected by ELISA; changes in oxidative stress levels (MDA, SOD, ROS) were analyzed using reagent kits; and the binding site between miR-32-3p and ATF4 was predicted through TargetScanHuman.; The miR-32-3p/ATF4 target relationship was validated using dual luciferase assays.</p> Results <p>ISO induced downregulation of miR-32-3p expression (<i>P &lt;</i> 0.0001), inhibition of AC16 cell proliferation (<i>P</i> &lt; 0.0001), upregulation of myocardial hypertrophy markers ANP and β-MHC, as well as increased levels of inflammatory cytokines IL-6, TNF-α, IL-1β and oxidative stress (all <i>P</i> &lt; 0.0001). When the miR-32-3p mimic was added, these changes were reversed. The dual luciferase assay confirmed that bioinformatics predicted miR-32-3p to directly target the 3’UTR of ATF4. The rescue experiment showed that overexpression of ATF4 eliminated the protective effect of miR-32-3p (<i>P</i> &lt; 0.05).</p> Conclusions <p>miR-32-3p alleviates ISO induced AC16 cell damage by targeting ATF4, regulating inflammation and oxidative stress.</p>