Background <p>Doxorubicin (DOX) can induce serious adverse reactions such as myocardial tissue injury, cardiac dysfunction, and heart failure in clinical application. Knockdown of thrombospondin 3 (THBS3) can protect the heart, but the mechanism of THBS3 in heart failure has not been reported.</p> Methods <p>Through the use of DOX to induce a cardiac injury model in elderly mice, AAV9-THBS3 and AAV9-sh-THBS3 were injected into the tail vein to overexpress and knockdown THBS3, while PI3K/AKT agonist 740Y-P and inhibitor LY294002 were injected intraperitoneally. THBS3 and the PI3K/AKT/eNOS pathway proteins of mouse heart tissues were detected by Western blot and immunofluorescence. ELISA and kits were used to detect the levels of biomarkers of myocardial injury. WGA, HE, and Masson staining were used to observe the pathological changes of heart tissues. DCFH-DA, transmission electron microscopy, Western blot, and TUNEL staining were used to detect mitochondrial dysfunction-related indicators.</p> Results <p>THBS3 expression was increased in DOX-induced heart tissue. Knockdown of THBS3 can raise the cross-sectional area of cardiomyocytes, reduce the swelling and vacuolation of cardiomyocytes and mitochondria, reduce the degree of blue-stained collagen and fibrosis, reduce the content of cTnT, CK-MB, BNP, LDH, and ROS, increase the protein levels of PPAR-α, PGC-1α, Sirt3, MFN2, and OPA-1, and reduce the level of apoptosis in heart tissue. Knockdown of THBS3 activates the PI3K/AKT/eNOS axis. After 740Y-P intervention, cardiac injury and mitochondrial dysfunction were further improved, while LY294002 aggravated cardiac injury and mitochondrial dysfunction.</p> Conclusion <p>Knockdown of THBS3 activates the PI3K/AKT/eNOS pathway to inhibit DOX-induced mitochondrial dysfunction in elderly mice, thereby improving heart damage caused by heart failure.</p> Highlights <p><OrderedList> <ListItem> <ItemNumber>1.</ItemNumber> <ItemContent> <p>THBS3 level is increased in the heart tissue of aged mice induced by doxorubicin (DOX).</p> </ItemContent> </ListItem> <ListItem> <ItemNumber>2.</ItemNumber> <ItemContent> <p>Knockdown of THBS3 attenuated DOX-induced cardiac injury in aged mice.</p> </ItemContent> </ListItem> <ListItem> <ItemNumber>3.</ItemNumber> <ItemContent> <p>Knockdown of THBS3 inhibited DOX-induced mitochondrial dysfunction in aged mice.</p> </ItemContent> </ListItem> <ListItem> <ItemNumber>4.</ItemNumber> <ItemContent> <p>Knockdown of THBS3 activates the PI3K/AKT/eNOS signaling pathway.</p> </ItemContent> </ListItem> </OrderedList></p>