Weightlessness is known to disrupt cellular homeostasis, partly by inducing oxidative stress and altering antioxidant defense systems. The present study aimed to investigate the effects of simulated weightlessness on antioxidant enzyme activity and lipid peroxidation in female Wistar rats. Twenty female rats were randomly assigned to two groups: a control group and a simulated weightlessness group. The hindlimb unloading model was employed for four weeks to mimic weightlessness conditions. Blood samples were collected at baseline, after two weeks, and after four weeks. Serum activities of superoxide dismutase and glutathione peroxidase, as well as the concentration of malondialdehyde, a biomarker of lipid peroxidation, were measured using commercial assay kits. In the HLU group, both superoxide dismutase and glutathione peroxidase activities significantly increased after two weeks, indicating a compensatory upregulation of the antioxidant defense system in response to oxidative stress. By the fourth week, superoxide dismutase activity had declined toward baseline levels, suggesting possible adaptation or exhaustion of the enzymatic defense, while glutathione peroxidase activity remained relatively elevated compared to the controls. Conversely, malondialdehyde levels were significantly elevated at both time points in the hindlimb unloading group and persisted throughout the experimental period, reflecting sustained lipid peroxidation and continuous oxidative stress. The control group showed no significant changes in any of the indices. These findings demonstrate that simulated weightlessness disrupts redox balance by inducing persistent oxidative stress in female rats. The observed compensatory but insufficient antioxidant response underscores the importance of considering antioxidant-based protective interventions during spaceflight and related biomedical conditions.