The spaceflight experiment was carried out using male C57BL/10J mice (8 weeks old at launch). Wild type mice (n=3) were launched by Space Shuttle Discovery and housed on the International Space Station (ISS) for 91 days. They returned to the Earth by Space Shuttle Atlantis. But only one mouse returned to the Earth alive. Whole brain was sampled from the mouse killed by inhalation of carbon dioxide at the Life Sciences Support Facility of Kennedy Space Center within 3-4 hours after landing. After the spaceflight experiment the on-ground experiment was also carried out at the Advanced Biotechnology Center in Genova Italy. A mouse with the same species sex and age was housed in mice drawer system (MDS) which was utilized for the spaceflight (SF) mice for 3 months as the ground control (GC). Another mouse was housed in normal vivarium cage as the laboratory control (LC). Amount of food and water supplementation and environmental conditions were simulated as the flight group. After 3 months brain was sampled from one mouse in group GC and LC respectively. Comprehensive analyses of gene expression were performed in the right brain. Total of 4,000 genes were analyzed. The expression levels of 60 genes significantly changed in response to SF compared with LC and/or GC. The 15 and 16 genes were up- (> 2 folds) and down-regulated (< 0.5 folds) respectively following SF vs. GC. The levels of 58 genes were significantly altered by housing in MDS in space and/or on the ground. Forty seven and 11 genes were significantly up- and down-regulated vs. LC. Twenty seven out of these genes responded to caging in MDS both in space and on the ground. Further 31 genes were influenced by housing in MDS on the Earth. Responses of the characteristics of brain to long-term gravitational unloading were investigated in mice.