In view of long-haul space exploration missions, the European Space Agency initiated the MicroEcological Life Support System Alternative (MELiSSA) project targeting the total recycling of organic waste produced by the astronauts into oxygen, water and food using a loop of bacterial and higher plant bioreactors. In that purpose, the a-proteobacterium, Rhodospirillum rubrum S1H, was sent twice to the International Space Station and was analyzed post-flight using a newly developed R. rubrum whole genome oligonucleotide microarray and high throughput gel-free proteomics with Isotope-Coded Protein Label technology. Moreover, in an effort to identify a specific response of R. rubrum S1H to space flight, simulation of microgravity and space-ionizing radiation were performed on Earth under identical culture set-up and growth conditions as encountered during the actual space journeys. Transcriptomic and proteomic data were integrated and permitted to put forward the importance of medium composition and culture set-up on the response of the bacterium to space flight-related environmental conditions. In addition, we showed for the first time that a low dose of ionizing radiation (2 mGy) can induce a significant response at the transcriptomic level, although no change in cell viability and only a few significant differentially expressed proteins were observed. From the MELiSSA perspective, we could argue the effect of microgravity to be minimized, whereas R. rubrum S1H could be more sensitive to ionizing radiation during long-term space exploration mission.