Background/Objectives: Stimulation of glucose uptake in response to ischemic stress is important for cardiomyocyte post-ischemic function and survival. In the diabetic myocardium chronically exposed to an excess of circulating lipids, this mechanism is impaired, making the myocardium more sensitive to ischemia–reperfusion injury (IRI). In vitro studies have shown that exposure to fatty acids (FAs) reduces basal and stimulated glucose uptake in cardiomyocytes. Preliminary results indicate reduced NAD+ levels and increased protein lysine acetylation in FA-exposed cardiomyocytes. This study aims to investigate whether intracellular NAD+ reduction is responsible for FA-induced increase in protein acetylation and impaired glucose uptake. Methods: Primary rat cardiomyocytes were chronically treated with the sirtuin deacetylase inhibitor nicotinamide (NAM) in absence of FAs to induce protein acetylation. Conversely, we replenished NAD+ concentration using nicotinamide riboside (NR) to induce protein deacetylation in FA-exposed cardiomyocytes. Results: Similar to FA exposure, NAM treatment increased protein acetylation and impaired metabolic-stress-stimulated glucose uptake in cardiomyocytes. In contrast, NR supplementation reduced protein acetylation and improved metabolic-stress-stimulated glucose uptake in FA-exposed cardiomyocytes. Neither NAM nor NR influenced insulin-stimulated glucose uptake. Both NAM and FAs induced hydroxyacyl-CoA dehydrogenase trifunctional enzyme subunit α (HADHA) acetylation on lysine residues K166 and K214 and enhanced palmitate oxidation. Conversely, NR treatment induced HADHA deacetylation and reduced palmitate uptake and oxidation in FA-exposed cardiomyocytes. Conclusions: In cardiomyocytes, protein hyperacetylation, resulting from either FA exposure or sirtuin inhibition, impairs metabolic-stress-stimulated glucose uptake and is associated with increased FA oxidation.