Hypoxic preconditioning is known to protect against various functional disorders, including skeletal muscle dysfunction. Na,K-ATPase, which plays an important role in adaptation to hypoxia, is critical for maintaining the performance of skeletal muscles, which co-express the α1 and α2 isozymes of the enzyme. Persistent dysfunction of α2 Na,K-ATPase isozyme is characteristic of the motor inactivity of skeletal muscles. In this study, we tested our hypothesis that mild hypoxia is able to improve Na,K-ATPase in disused soleus muscle. Rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Then, 18 h after control or HH conditions, rats were subjected to 6 h of hindlimb suspension (HS), a model of skeletal muscle disuse. Isolated soleus muscles were tested. HH itself stably increased the α2 Na,K-ATPase isozyme membrane abundance and its electrogenic activity. These effects were accompanied by a decrease in serum level of circulating endogenous ouabain, a specific ligand for Na,K-ATPase. HS itself caused loss of electrogenic activity of α2 Na,K-ATPase, but pretreatment with HH protected against this impairment. This protective effect was accompanied by an increase in the α2 Na,K-ATPase membrane abundance without a change in total protein content, suggesting an increase in the α2 Na,K-ATPase traffic from the intracellular pool to the sarcolemma; serum ouabain level was significantly increased. We suggest that these findings open a new field for further studies and may have therapeutic implications for disuse-induced skeletal muscle pathology.