Astronauts on board the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. One of the most critical and sensitive districts of their organism is the eye, and in particular the retina, so that more than half of them develops a complex of alterations designated as Spaceflight Associated Neuro-ocular Syndrome. We explored the cellular and molecular effects induced on human retinal pigment ARPE-19 cell line by their transfer to and three days stay on board the ISS in the context of an experiment funded by the Agenzia Spaziale Italiana (ASI). Treatment of cells on board ISS with the well-known bioenergetic, antioxidant and antiapoptotic coenzyme Q10 was also evaluated. In the ground control experiment the cells were exposed to the same conditions as on the ISS, except for microgravity and radiation. Transfer of ARPE-19 retinal cells to the ISS and their living on board for three days did not impact on cell viability or apoptosis but induced cytoskeleton remodeling consisting in vimentin redistribution from the cellular boundaries to the perinuclear area, underlining the collapse of the network of intermediate vimentin filaments under unloading conditions. Morphological changes endured by ARPE-19 cells grown on board the ISS were associated with changes in the transcriptomic profile related to cellular response to space environment, and were consistent with cell dysfunction adaptations. In addition, results obtained from ARPE-19 cells treated on board ISS with coenzyme Q10 showed its potential ability to increase cell resistance to damaging insults.