<p>Drug-induced mitochondrial toxicity is a major contributing factor to cardiotoxicity, which can cause drug attrition and adverse cardiac events. To assess the toxicity of anti-inflammatory agents, we used adult human primary cardiomyocytes (hPCMs) to screen 18 clinically available anti-inflammatory drugs in a high-content manner, and revealed widespread mitochondrial dysfunction without affecting cell viability. Nabumetone, a representative nonsteroidal anti-inflammatory drug with profound mitochondrial toxicity, induced mitochondrial fission, inhibited mitophagy, and impaired both electrophysiological and metabolic functions in adult hPCMs. Mechanistically, we uncovered that nabumetone (Nab) exerted its toxic effects through the prostaglandin E2- E-type prostanoid receptor 4 (PGE₂-EP₄) pathway, which was essential for its anti-inflammatory functions. To find an alternative route to ameliorate mitochondrial damage, we identified SIRT3 as a downstream target of nabumetone. Its mRNA, protein, and activity levels were significantly reduced upon nabumetone treatment. SIRT3 activator honokiol exhibited protective potential against NSAID-induced mitochondrial toxicity both in hPCMs and in nabumetone-treated mice. Finally, through screening mitochondrial liability in various common cardiomyocyte models, we identified mitochondrial abundance as an important determinant of the sensitivity of cells towards mitochondrial toxicants. Our study demonstrates the vast presence of mitochondrial dysfunction in human adult cardiomyocytes imposed by clinically used anti-inflammatory drugs, and identified both toxicity and protective pathways that may serve future therapeutic purposes.</p>