Abstract Body: During ischemic cardiac injury, cardiac fibroblasts (CFs) transition from resident CF to a myofibroblast and undergo a metabolic shift from oxidative metabolism to anaerobic glycolysis. It has previously been shown that alterations in metabolism can play a significant role in determining cell identity and function, but this idea has been minimally explored in CFs. The Hexosamine Biosynthesis Pathway (HBP) is an accessory pathway to glycolysis and interestingly, the rate-limiting enzyme, glutamine-fructose-6-phosphate transaminase 2 (Gfpt2) increases significantly in CFs following a myocardial infarction (MI). This suggests a possible role for Gfpt2 and the HBP in CFs that has not been previously studied. We hypothesized that Gfpt2 plays an essential role in the CF to myofibroblast transition and in the CF response to ischemic injury through metabolic reprogramming. To study Gfpt2 we utilized adult mouse cardiac fibroblasts in vitro to perform functional studies, bulk RNA sequencing and untargeted metabolomics. Following Gfpt2 knockdown, bulk RNA sequencing demonstrated significant changes to gene expression including an upregulation of extracellular matrix related pathways suggesting a shift towards a more activated/myofibroblast-like state. Additionally, it suggested an overlap between pathways affected by Gfpt2 knockdown and pathways affected during CF activation suggesting that Gfpt2 may regulate a portion of this process. Untargeted metabolomics analysis further supported that there are shared pathways between Gfpt2 knockdown and CF activation. A significant shared pathway was glutathione metabolism which is historically known for its role in reduction-oxidation reactions and in the processing of reactive oxygen species. Ongoing work continues to test how glutathione can affect CF activation and on further defining the role of Gfpt2 in this process. In conclusion, we have demonstrated how Gfpt2 modulates CF activation and have identified glutathione as a downstream pathway that may affect the CF ischemic injury response.