Abstract Body: Introduction: Doxorubicin (DOX) is a first line chemotherapeutic agent. However, its clinical utility is limited by off-target side effects. Notably, DOX causes cardiotoxicity in a dose-dependent manner. While the molecular underpinnings of DOX induced cardiotoxicity (DIC) are incompletely understood, excess reactive oxygen species (ROS) generation and ensuing mitochondrial dysfunction are thought to contribute to this pathology. Whether previously identified cardioprotective stress-response pathways, such as the mitochondrial unfolded protein response (UPRmt), are operative in mitigating DOX cardiotoxicity remains unknown. Furthermore, whether potential DOX induced UPR^mt signaling is orchestrated by the transcription factor, ATF5, is also unknown. Hypothesis: We hypothesize that ATF5 mediates DOX induced UPR^mt signaling in cardiomyocytes, as a means of attenuating cardiotoxicity. Method: We performed time course experiments using both in-vitro (AC-16 human cardiac cells) and in-vivo (C57BL/6 mice) models of DIC, to elucidate UPR^mt signaling patterns within cardiac cells and mouse hearts exposed to DOX. Stable overexpression of ATF5 in AC-16 cells via lentiviral transduction was used to investigate ATF5's role in orchestrating UPR^mt signaling in cardiac cells exposed to DOX. Results: In AC-16 cells, DOX upregulated UPR^mt associated gene transcripts in a dose and time-dependent manner. A significant increase in transcript levels of AFG3L2 (p=0.0008), CLPP (p=0.0112), and CLPX (p=0.0043), were observed between vehicle and 1µM DOX treated cells at 12 hrs. post treatment. These results were recapitulated in-vivo, whereby hearts of mice treated with 10mg/kg of DOX for 24 hrs. exhibited significant increases in CLPX, HSPA9 and LONP1 transcripts as compared to vehicle treated control mice. Protein levels of CLPP and CLPX were also significantly increased. UPR^mt signaling was enhanced in DOX-treated ATF5 overexpression AC-16 cells compared with DOX-treated control cells, leading to a significant reduction in the apoptotic index and improved cell viability. Conclusion: These data highlight ATF5 mediated UPR^mt signaling as a cardioprotective response within cells of the heart exposed to DOX. Future efforts will explore pharmacologic and genetic manipulation of the UPR^mt to ameliorate DIC.