The mammalian brain stores glucose, the main energy substrate from the blood circulation, in the form of glycogen. In the rodent brain, the cerebellum stores relatively large amounts of glycogen, yet the cellular and subcellular distribution of glycogen has not been described in detail. Using monoclonal antibodies against glycogen, we investigated the glycogen distribution in the mouse cerebellar cortex. We found a dominant presence of glycogen in molecular layer Bergmann glia (BG) processes. Additionally, glycogen was observed in Purkinje cells (PCs), the principal neurons of the cerebellar cortex. To address the functional significance of cerebellar glycogen, we examined behavioural phenotypes in mice that lack glycogen synthase 1 (Gys1) in cerebellar astrocytes (which include BG) or PCs using a Gys-1 floxed mouse line. Gys1 deficiency in PCs (by L7-Cre transgenic mouse or AAV-L7-Cre) or AAV-GFAP-Cre- infected cells alone did not result in a notable phenotype, however, Gys1 deficiency in both cell types induced ataxia. Histological examination showed that the dual cell-type gys1 deficiency ablated PCs. Our observations elucidate a critical involvement of glycogen metabolism for the normal operation of the cerebellum.