The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH. Display omitted •HK1 mitochondrial dissociation results in GAPDH inhibition and increased PPP•Cytosolic HK1 increases macrophage-mediated inflammatory cytokine production•HK1 associates with S100A8/A9 and attenuates GAPDH activity through nitrosylation•Diabetic and aging immune cells show cytosolic HK1 and reduced GAPDH activity De Jesus et al. describe how HK1 subcellular localization regulates the metabolic fate of its substrate, G6P, between catabolic and anabolic metabolism through inhibition of GAPDH. Immune cells from models of aging and diabetes show higher levels of cytosolic HK1 and increased inflammation.