Objective With China’s manned spaceflight project entering the medium- and long-term flight stages, the effects of the weightless environment on cognitive function during long-term stays in space has become an important challenge. This study aimed to explore the influence of a weightless environment on learning and memory ability in mice, and to investigate its potential molecular mechanism by establishing a simulated space-weightlessness model on the ground. Methods Male ICR mice were used to establish a simulated space-weightlessness model by tail-suspension method, and the intervention lasted for 4 weeks. Changes in learning and memory ability were evaluated by behavioral tests including the Y-maze, new object-recognition, Morris water maze, step-down test, and shuttle-box test. The H₂O₂ and malondialdehyde (MDA) in serum were detected by enzyme-linked immunosorbent assay, and markers of oxidative stress (nuclear factor erythroid 2-related factor 2(Nrf2) / heme oxygenase-1 (HO-1) pathway), inflammation ( interleukin ( IL)-33, cyclooxygenase-2 ( COX-2)), apoptosis ( Caspase-3, Caspase-8,Bcl2 / Bax), and mitochondrial function (mitochondrial transcription factor A (mtTFA), phospho-mechanistic target of rapamycin (P-mTOR) / mTOR, SirT1, SirT3) were detected in the hippocampus by Western blot. Results After 4 weeks of simulated weightlessness, the discrimination index of mice in the new object-recognition experiment was reduced compared with the control group (P<0. 05), markedly prolonged escape latency in the Morris water maze (P<0. 01), extremely significantly reduced time spent in the safe zone in the step-down test (P<0. 001), and notably decreased numbers of active avoidance responses in the shuttle-box test (P<0. 01, P<0. 001, P<0. 05). The H₂O₂ and MDA contents in brain tissue were increased (P<0. 05). The relative expression levels of Nrf2 and HO-1 protein in the hippocampus were decreased in the simulated-weightlessness mice ( P<0. 001, P<0. 05). The relative expression levels of the inflammatory-related proteins IL-33 and COX-2 were increased (P<0. 05, P<0. 001); the pro-apoptosis-related proteins Bax, Caspase-3, and Caspase-8 were increased (P<0. 01); the anti-apoptosis protein Bcl2 was decreased ( P<0. 01); and the relative expression levels of the mitochondrial function-related proteins mtTFA, P-mTOR/ mTOR, SirT1, and SirT3 were decreased ( P<0. 001, P<0. 05, P<0. 01, P<0. 001) in simulated-weightlessness mice. Conclusions Simulated weightlessness can lead to a significant decline in learning and memory ability in mice, and its mechanism may be related to multiple pathophysiological processes such as increased oxidative stress, activation of the inflammatory response, and increased apoptosis and mitochondrial dysfunction in the hippocampus. This study provides an experimental basis for understanding the pathogenesis of cognitive dysfunction in a space-weightlessness environment, and lays a theoretical foundation for developing targeted protective measures.