Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts towards faster muscle fiber types. To identify changes in gene expression which may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11 day, 19 hour STS-108 shuttle flight and from normal gravity controls. Spaceflight data were also compared to the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 hours of reloading to mimic the time between landing and sacrifice of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, while reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the PI3 kinase regulatory subunit p85, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase while mRNA levels of the myostatin inhibitor FSTL3 tended to decrease in response to spaceflight. In addition, mRNA levels of the slow-oxidative fiber associated transcriptional co-activator peroxisome proliferator associated receptor-(PPAR) gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.