Exposure to long-duration microgravity leads to ocular changes in astronauts, manifested by a variety of signs and symptoms during spaceflight that in some cases persist after return to Earth. These morphological and functional changes are only partly understood and are of occupational health relevance. To investigate further into the molecular basis of the changes occurring in ocular tissue upon exposure to spaceflight, eyes were collected from female C57BL/6 mice flown on STS-135 (FLT) on landing day or from their ground control counterparts maintained at similar conditions within the Animal Enclosure Module (AEM). One eye was fixed for histological sectioning while the contralateral eye was dissected to isolate the retina for gene expression profiling. 8-hydroxy-deoxyguanosine (8OHdG) staining showed a statistically significant increase in the inner nuclear layer of FLT samples compared to AEM. Gene expression analysis in isolated retina identified 139 differentially expressed genes in FLT compared to AEM control samples. The genes affected were mainly involved in pathways and processes of endoplasmic reticulum (ER) stress, inflammation, neuronal and glial cell loss, axonal degeneration, and herpes virus activation. These results suggest a concerted change in gene expression in the retina of mice flown in space, possibly leading to retinal damage, degeneration, and remodeling.