Spaceflight exploration presents environmental stressors including microgravity-induced cephalad fluid shift and radiation exposure. Ocular changes leading to visual impairment in astronauts are of occupational health relevance. The effect of this complex environment on ocular morphology and function is poorly understood. Female 10-12 week-old BALB/cJ mice were assigned to a flight (FLT) group flown on shuttle mission STS-133, Animal Enclosure Module ground control group (AEM), or vivarium-housed (VIV) ground controls. Eyes were collected at 1, 5, and 7 days after landing and were fixed for histological sectioning. The contralateral eye was used for gene expression profiling by RT-qPCR. Sections were visualized by hematoxylin/eosin stain and processed for 8-hydroxy-2'-deoxyguanosine (8-OHdG), caspase-3, and glial fibrillary acidic protein (GFAP) and β-amyloid double-staining. 8-OHdG and caspase-3 immunoreactivity was increased in the retina in FLT samples at return from flight (R+1) compared to ground controls, and decreased at day 7 (R+7). Β-amyloid was seen in the nerve fibers at the post-laminar region of the optic nerve in the flight samples (R+7). Expression of oxidative and cellular stress response genes was upregulated in the retina of FLT samples upon landing, followed by lower levels by R+7. These results suggest that reversible molecular damage occurs in the retina of mice exposed to spaceflight and that protective cellular pathways are induced in the retina and optic nerve in response to these changes.