Gangliosides, as major lipid raft (LR) constituents, provide an optimal milieu for the function and activity of various membrane proteins. One example is the Na+,K+-ATPase (NKA) vital for cell ion homeostasis. Aiming to investigate effects of gangliosides on NKA gene and protein expression, enzyme activity, and submembrane localization, we analyzed cortices and cerebella derived from a mouse model with impaired synthesis of gangliosides (St8sia1 null) in comparison with wild type (WT) mice. Gene expression of six NKA isoforms was determined by qRT-PCR, while NKA protein expression was analyzed by Western blotting and immunohistochemistry. NKA activity was measured spectrophotometrically. LR and non-raft (nLR) fractions were isolated by ultracentrifugation in sucrose gradients, and analyzed by Western blotting. We show that NKA activity is lower in the cortices of null mice compared to the WTs. In accordance, total NKA protein amount was lower in null mice cortices compared to WTs. Immunohistochemistry revealed a lower intensity of NKA staining in null mice's cortices and cerebellar molecular and granular layers than in WT mice. However, we found higher gene expression of Atp1a1 and Atp1a2 in both cortices and cerebella, respectively. Analysis of submembrane localization has shown a higher amount of NKA to be positioned within the LRs of cortices than those derived from the cerebella. Our findings indicate an intricate relationship between gangliosides and NKA demonstrating that all levels of NKA expression are influenced by changed ganglioside environment, affecting its micropositioning and function which may ultimately lead to a disbalance in cellular ion homeostasis.