Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that can ferment cellulose to ethanol in a consolidated bioprocess, and it is a candidate industrial biocatalyst for cellulosic fuel ethanol production. C. thermocellum is relatively sensitive to ethanol compared to current industrial yeast. Previous studies have investigated the membrane and protein composition of wild-type and ethanol tolerant strains and we have resequenced the genomes for such strains. However, relatively little is known about its physiological response and the coordinate regulatory response to ethanol stress. In this study, wild-type C. thermocellum cultures were grown to mid-exponential phase and then either shocked with the addition of ethanol to a final concentration of 0.5% or were untreated. Samples were taken pre-shock and 2, 12, 30, 60, 120, 240 min post-shock for multiple systems biology analyses. The addition of ethanol dramatically decreased C. thermocellum growth and the final cell density was approximately half of the control fermentations, with concomitant reductions in substrate consumption rates in the treated cultures. The final extracellular ethanol, lactate and acetate concentrations were similar at between in the control and treated cultures at the end of the experiment. The response of C. thermocellum to ethanol was dynamic and involved about 500 genes that were significantly and differentially expressed between the different conditions over time, which represented every functional category. Cellobiose was accumulated within the ethanol-shocked C. thermocellum cells, as well as the sugar phosphates such as fructose-6-P and glucose-6-P. The comparison between intracellular metabolites, proteomic and transcriptomcs profiles will be discussed.