Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight host factors that could contribute to oral diseases such as caries and periodontitis. However the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study High Aspect Ratio Vessel (HARV) S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles H2O2 resistance and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated >= 2-fold and 94 genes down-regulated >= 2-fold during simulated microgravity HARV growth. These included a number of genes located on extrachromosomal elements as well as genes involved in carbohydrate metabolism translation and stress responses. Collectively these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions. Overall design: Differential gene expression was compared between RNA from S. mutans grown in normal gravity HARVs (n=3 independent cultures) and RNA from S. mutans grown in simulated microgravity HARVs (n=3 independent cultures)