Hexokinase (HK)(E.C.188.8.131.52) is the enzyme responsible for catalyzing the first step of glucose metabolism, the phosphorylation of glucose to glucose-6-phosphate (G6P). The present study investigated HK from tail muscle of the freshwater crayfish Oconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20 h anoxic). Evidence indicated that HK was converted to a low phosphate form under anoxia. ProQ Diamond phosphoprotein staining showed a 39% higher bound phosphate content on aerobic HK compared with the anoxic form, yet treatment of aerobic HK with treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1+PP2A, PP2B, and PP2C) yielded no significant changes in kinetic parameters. By contrast, investigation of the stability and bound fractions of aerobic verses anoxic HK yielded stark differences in both susceptibility to urea denaturation and subsequent proteolytic cleavage, as well as a decrease in the amount of enzyme in the bound state. The physiological consequence of anoxiainduced HK dephosphorylation may be to stabilize and release HK during anoxia, increasing the glycolytic capacity of the animal.