Na+,K+-ATPase is a membrane protein which plays a key role in the maintenance of ion homeostasis that is necessary to neuronal excitability, secondary transport and neurotransmitter uptake. Mild hyperhomocysteinemia leads to several clinical manifestations and particularly cerebral diseases; however, little is known about the mechanisms of homocysteine on cerebral Na+,K+-ATPase. In the present study, we investigated the effect of mild hyperhomocysteinemia on the activity, the immunocontent of catalytic subunits (α 1, α 2, and α 3) and the gene expression of this enzyme. We used the experimental model of mild hyperhomocysteinemia that was induced by homocysteine administration (0.03 μmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Results showed that mild hyperhomocysteinemia significantly decreased the activity and the immunocontent of the α 1 and α 2 subunits of the Na+,K+-ATPase in cerebral cortex and hippocampus of adult rats. On the other hand, we did not observe any change in levels of Na+,K+-ATPase mRNA transcripts in such cerebral structures of rats after chronic exposure to homocysteine. The present findings support that the homocysteine modulates the Na+,K+-ATPase and this could be associated, at least in part, with the risk to the development of cerebral diseases in individuals with mild hyperhomocysteinemia.