Abstract: This study employs bioinformatics methods to analyze and compare the effects of neutron radiation and microgravity environments on retinal protein expression in mice, providing a biological basis for understanding the mechanisms of retinal damage induced by space environments. Furthermore, it offers insights for risk assessment and protective measures related to space environments. We obtained differential expression data of retinal proteins in mice exposed to neutron radiation and microgravity environments. Various bioinformatics methods, including GO and KEGG enrichment analyses, PPI network construction and module analysis, and Hub protein screening and analysis, were employed to compare the effects of neutron radiation and microgravity on retinal protein differential expression. The results show that there were differences in the most significantly enriched functions. Neutron radiation inducement primarily enriched in functions such as “response to xenobiotic stimulus”, “actin filament”, and “identical protein binding”. Microgravity inducement enriched in functions such as “lens development in camera-type eye”, “mitochondrion”, and “structural constituent of eye lens”. The KEGG analysis showed that the changes in the “Motor proteins” pathway were consistent under both neutron radiation and microgravity inducement. In addition, neutron radiation inducement also enriched in the “Focal adhesion” and “Regulation of actin cytoskeleton” pathways, while microgravity inducement also enriched in the “Cardiac muscle contraction” pathway. The Hub proteins and Biological Process (BP) of the most significant modules were different. The most significant BP under neutron radiation inducement was “muscle contraction”, while under microgravity inducement, “camera-type eye development” was the most significant. Under different inducement, the expression trends of proteins PMEL, PTN, TPM1, and RAB27A were consistent. However, PDPN showed an opposite change. The results suggest that retinal proteins respond differently to neutron radiation and microgravity stimuli. These findings have the potential to elucidate the mechanisms of retinal damage caused by space radiation or microgravity and provide a reference for developing targeted protective measures.