Background: Cardiotoxicity of doxorubicin (DOX) is mediated through mitochondrial perturbations. SIRT1, an NAD + -dependent deacetylase, has been reported to promote cellular adaptation to metabolic imbalances in part via mitochondrial biogenesis. Hypothesis: SIRT1 in the cardiomyocyte counteracts DOX cardiotoxicity in vivo. Methods: Tamoxifen (Tam)-inducible cardiomyocyte-specific SIRT1 knockout mice (SIRT1 cKO) were used. At 2 months of age, mice received Tam to induce knockout, and mice without Cre recombinase served as wild type (WT). Both groups of mice were treated with DOX (4 IP injections of 5 mg/kg/week) starting at 3 months of age. Echocardiography was performed before and after DOX treatment. Cardiac tissues were sampled after the final DOX injection. H9c2 cardiomyocytes were used for in vitro analysis. Results: At baseline, there were no differences in left ventricular (LV) dimension, thickness, fractional shortening (FS), and ejection fraction (EF) between the groups. DOX significantly reduced FS, EF, and LV thickness in both groups of mice. However, FS and EF after DOX injections were lower in SIRT1 cKO than in WT (FS, 26% vs. 30%; EF, 59% vs. 65%, P<0.05). ANP and BNP mRNA levels were also higher in SIRT1 cKO (2.4-fold and 2.4-fold, respectively, P<0.05), confirming worsening of cardiac function. Compared with WT, SIRT1 cKO showed reduced cardiac mitochondrial content determined by qPCR analysis of mitochondrial DNA level (-16%, P<0.05). Unexpectedly, mRNA levels of nuclear-encoded mitochondria genes (Cox5b, Atp5a1, and citrate synthase) were not altered in the SIRT1 cKO heart, indicating preserved mitochondrial biogenesis in cKO. Instead, mRNA levels of parkin (+43% P<0.05), PINK1 (+18%), and Bnip3 (+40%) were higher in SIRT1 cKO than in WT, suggesting enhanced mitochondrial degradation via mitophagy. Finally, SIRT1 knockdown in H9c2 cells enhanced DOX (10 μM, 24 h)-induced cell death examined by LDH release compared with control cells (13.2±1.4% vs. 8.6±0.7%, P<0.05). Conclusions: The results suggested that SIRT1 in the cardiomyocyte counteracts DOX cardiotoxicity by preserving mitochondrial content in vivo. Enhanced degradation, but not impaired biogenesis, may be involved in mitochondrial depletion in SIRT1 cKO.