Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat Pseudomonas aeruginosa. Overall P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA) involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions were hypothesized to be at the origin of these observations. Collectively our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen results that are of importance for infectious disease risk assessment and prevention both during spaceflight missions and in the clinic. This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end P. aeruginosa cultures were grown in space and the expression profile was compared with ground control samples (both in biological triplicate). Two RWV samples also examined (did not re-analyze them only compared the outputs).