Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol in winter that serves as an antifreeze. Liver glycogen is a source of glycerol during the early stages of glycerol accumulation while dietary glucose and amino acids are essential to maintain rates of glycerol synthesis. Here we report rates of glycerol and glucose production by isolated hepatocytes. Cells from fish held at 0.4°C to -1.5°C and incubated at 0.4°C were metabolically quiescent with negligible rates of glycerol or glucose production. Hepatocytes isolated from fish maintained at 8°C and incubated at 8°C produced glucose but not glycerol. Glycerol production was activated in cells isolated from 8°C fish and incubated at 0.4°C without substrate or when glucose, aspartate or pyruvate was available in the medium. Incubation at 0.4°C without substrate resulted in similar molar rates of glucose and glycerol production in concert with glycogen mobilization. Glycogenolysis and glycerol production were associated with increases in total in vitro activities of glycogen phosphorylase and glycerol 3-phosphate dehydrogenase. Maximal in vitro activities of hexokinase (HK) and glucokinase were not influenced by temperature but high activities of a low-K_m HK may serve to redirect glycogen derived glucose to glycolysis as opposed to releasing it from the cells. Rates of glycerol production were not enhanced in cells from fish held at 8°C and incubated at 0.4°C with adrenergic or glucocorticoid stimulation. As such, low temperature alone is sufficient to activate the glycerol production mechanism and results in a shift from glucose to a mix of glucose and glycerol production.