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Background/Aims: Microgravity [μg] has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of μg on human adult retinal epithelium (ARPE-19) cells is unknown. Methods: In this study we investigated the influence of simulated μg (s-μg: 5 and 10 days (d)), using a Random Positioning Machine (RPM), on ARPE-19 cells. We performed phase-contrast/ fluorescent microscopy, qRT-PCR, Western blotting and pathway analysis. Results: Following RPM-exposure a subset of ARPE-19 cells formed multicellular spheroids (MCS), whereas the majority of the cells remained adherent (AD). After 5d, alterations of F-actin and fibronectin were observed which reverted after lOd-exposure, suggesting a time-dependent adaptation to s-μsg. Gene expression analysis of 12 genes involved in cell structure, shape, adhesion, migration, and angiogenesis suggested significant changes after a lOd-RPM-exposure. 11 genes were down-regulated in AD and MCS lOd-RPM-samples compared to lg, whereas FLK1 was up-regulated in 5d- and lOd-RPM-MCS-samples. Similarly, TIMP1 was up-regulated in 5d-RPM-samples, whereas the remaining genes were down-regulated in 5d-RPM-samples. Western blotting revealed similar changes in VEGF, (3-actin, laminin and fibronectin of 5d-RPM-samples compared to lOd, whereas different alterations of p-tubulin and vimentin were observed. The pathway analysis showed complementing effects of VEGF and integrin β-1. Conclusions: These findings clearly show that s-μsg induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of ARPE-19, in addition to changes in cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis. [ABSTRACT FROM AUTHOR]