Rationale: Anthracyclines such as doxorubicin are among the most effective chemotherapeutic agents; however, their use leads to frequent cardiovascular complications, including heart failure, due to the activation of multiple cardiomyocyte injury pathways. Insulin-like growth factor binding protein-3 (IGFBP3) is strongly upregulated in cardiomyocytes after administration of doxorubicin. However, its role in doxorubicin-induced cardiomyocyte apoptosis and survival remains controversial. Methods and Results: We demonstrate that IGFBP3 is strongly induced by doxorubicin in neonatal ventricular myocytes (NRVMs) 12 hours after doxorubicin treatment by RT-PCR (4.2 fold vs. control, n=3, P<0.05) and Western blot. Adenoviral overexpression of IGFBP3 in NRVMs leads to a significant reduction of phosphorylated H2A histone (γH2AX) after doxorubicin treatment, a hallmark of DNA double-strand breaks (DSBs), as well as topoisomerase IIβ, a key mediator of doxorubicin induced DNA DSBs. Conversely, silencing of IGFBP3 with siRNA leads to a significant increase of γH2AX expression in NRVMs. Adenoviral shRNA silencing of IGFBP3 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) further demonstrates a 44% and 52% decrease in contraction magnitude and velocity in response to doxorubicin compared with scramble shRNA (scramble shRNA, n=5, shIGFBP3, n=3, P<0.05). Immunofluorescent imaging and cell fractionation assays conducted in NRVMs indicate that doxorubicin initiates IGFBP3 nuclear translocation, suggesting an active role in the cardiomyocyte response to doxorubicin-induced cell injury. Next generation sequencing of hiPSC-CMs treated with doxorubicin reveals that overexpression of IGFBP3 leads to upregulation of cardiomyocyte survival pathways, including TNF receptors without cytoplasmic death domain (TNFRSF10C and D), BCL2 family gene BCL2L1, the p53 target ACER2, the p53 co-factor FBXW7, the β-catenin mediator TBX3 and the ataxia telangiectasia mutated (ATM) activator ZNF423. Conclusion: Our findings indicate that IGFBP3 is strongly induced in cardiomyocytes by doxorubicin treatment. Overexpression of IGFBP3 attenuates doxorubicin induced DNA DSBs and promotes pro-survival signaling pathways.