Abstract Body (Do not enter title and authors here): The co-chaperone BAG3 is critical for protein quality control at the cardiac sarcomere. BAG3 binds to Hsp70 and coordinates the assembly of the CASA (chaperone-assisted selective autophagy) complex, thus supporting proteostasis and cardiomyocyte contractility. BAG3 mutations and/or decreased BAG3 levels are associated with cardiomyopathies, whereas BAG3 overexpression rescues ventricular function after myocardial infarction in mice. Despite BAG3’s promise as a therapeutic target, the mechanisms underlying BAG3 regulation are largely unresolved. Here, we investigate the mechanisms of BAG3 downregulation after stress. We found that BAG3 protein is reduced in human dilated cardiomyopathy hearts compared to non-failing hearts, yet there is an increase in bag3 mRNA transcript, suggesting BAG3’s downregulation in heart disease may be controlled post-transcriptionally. To identify these post-transcriptional pathways, we subjected neonatal rat ventricular myocytes (NRVMs) to prolonged hypoxia-reoxygenation (H/R) stress, which recapitulated the decrease in BAG3 levels observed in human heart disease. Notably, disrupting Hsp70 binding to BAG3 in NRVMs via the drug JG-98 decreases BAG3’s half-life by ~90%, suggesting that Hsp70 protects BAG3 from degradation. Loss of Hsp70-mediated protection could contribute to declining BAG3 levels, so we quantified Hsp70 abundance after H/R stress in NRVMs, finding no significant change. We also found that overexpressing inducible Hsp70 did not rescue BAG3 levels. To examine BAG3 regulation in vivo, we subjected wildtype mice to ischemia-reperfusion injury. After 24 hours, male mice had no change in Hsp70 abundance in the left ventricle, whereas Hsp70 was significantly upregulated in female mice. Despite this difference in Hsp70, BAG3 levels were decreased by ~20% in both sexes. Thus, our in vivo and in vitro data both suggest that BAG3 downregulation is not caused by loss of Hsp70 binding/protection. Interestingly, the decline in full-length BAG3 (85 kDa) was accompanied by an increase in a BAG3 cleavage product at 74 kDa. We analyzed this product via mass spectrometry, discovering that it lacks a third of the WW domain, which is involved in autophagy. In future experiments, BAG3 cleavage will be explored as a potential mechanism of BAG3 loss. Such mechanisms will provide insight into how to maintain BAG3 levels, and thus cardiac function, during stress.