Background/Objectives: Ischemia-reperfusion injury (IRI) contributes significantly to cardiomyocyte death following myocardial infarction, largely through sustained inflammatory signaling. This study aimed to identify stress-responsive deubiquitinating enzymes involved in cardiomyocyte injury under ischemic stress. Methods: HL-1 cardiomyocytes were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), followed by transcriptomic analysis, genetic manipulation of Usp31, biochemical assessment of p65 ubiquitination, NF-κB reporter assays, and in vitro deubiquitinase activity assays using sodium tanshinone IIA sulfonate (STS). Results: Usp31 was persistently upregulated in HL-1 cardiomyocytes after OGD/R. Depletion of Usp31 improved cell viability and reduced lactate dehydrogenase (LDH) release under OGD/R conditions. Mechanistically, Usp31 stabilized p65 and supported NF-κB activation, whereas Usp31 deficiency increased p65 ubiquitination and limited NF-κB transcriptional activity. STS inhibited recombinant Usp31 enzymatic activity under the experimental conditions tested and reduced Usp31-associated NF-κB activation and cardiomyocyte injury in cellular assays. Conclusions: These findings identify Usp31 as a candidate regulator of NF-κB-associated inflammatory responses in cardiomyocytes and support STS as a pharmacological modulator of Usp31-associated signaling with potential relevance to myocardial ischemic injury.