Microgravity has a profound impact on the physiology of pathogenic microbes; however, its effects on mutualistic microbes are relatively unknown. To examine the effects of microgravity on those beneficial microbes that associate with animal tissues, we used the symbiosis between the bobtail squid Euprymna scolopes and a motile, luminescent bacterium, Vibrio fischeri as a model system. Specifically, we examined the role of Hfq, an RNA-binding protein known to be an important global regulator under space flight conditions, in the squid–vibrio symbiosis under simulated microgravity. To mimic a reduced gravity environment, the symbiotic partners were co-incubated in high-aspect-ratio rotating wall vessel bioreactors and examined at various stages of development. Results indicated that under simulated microgravity, hfq expression was down-regulated in V. fischeri. A mutant strain defective in hfq showed no colonization phenotype, indicating that Hfq was not required to initiate the symbiosis with the host squid. However, the hfq mutant showed attenuated levels of apoptotic cell death, a key symbiosis phenotype, within the host light organ suggesting that Hfq does contribute to normal light organ morphogenesis. Results also indicated that simulated microgravity conditions accelerated the onset of cell death in wild-type cells but not in the hfq mutant strains. These data suggest that Hfq plays an important role in the mutualism between V. fischeri and its animal host and that its expression can be negatively impacted by simulated microgravity conditions.