The creatine/phosphocreatine shuttle system, as catalysed reversibly by creatine kinases, is thought to be essential for the storing and buffering of high phosphate-bound energy in tissues with high energy demand. In the present study, we aimed to clarify the cellular system of creatine biosynthesis and its energy metabolism in the mouse brain by immunohistochemistry for creatine biosynthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), ubiquitous mitochondrial creatine kinase (uCK-Mi) and brain-type cytoplasmic creatine kinase (CK-B). GAMT was expressed highly in oligodendrocytes and olfactory ensheathing glia and moderately in astrocytes, whereas GAMT was very low in neurons and microglia. By contrast, uCK-Mi was expressed selectively in neurons and localized in their mitochondria in dendrites, cell bodies, axons and terminals. The distinct and almost complementary distribution of GAMT and uCK-Mi suggests that the creatine in neuronal mitochondria is derived not only from the circulation, but also from local glial cells associated with these neuronal elements. By contrast, CK-B was selective to astrocytes among glial populations, and was exclusive to inhibitory neurons among neuronal populations. Interestingly, these cells with high CK-B immunoreactivity are known to be highly resistant to acute energy loss, such as hypoxia and hypoglycemia. Considering that phosphocreatine generates ATP much faster than the processes of glycolysis and oxidative phosphorylation, the highly regulated cellular expressions of creatine biosynthetic and metabolic enzymes suggest that the creatine/phosphocreatine shuttle system plays a role in brain energy homeostasis through a novel neuron–glial relationship.