The anaerobic thermophilic bacterium Clostridium thermocellum ferments cellulose directly to ethanol and other metabolic products using its multi-enzyme cellulosome complex without the need for external cellulase addition. Elimination of cellulase production step consolidates the cellulosic ethanol production process significantly reducing costs. In this study, we used microarray technology to probe the genetic expression of C. thermocellum ATCC 27405 during cellulose and cellobiose fermentation. We also used multidimensional LC-MS/MS technology and ¹⁵N-metabolic labeling strategy to quantify changes in cellulosomal proteins in response to various carbon sources (cellobiose, amorphous/crystalline cellulose (avicel) and combinations of avicel, pectin and xylan). Transcriptomic analysis involved a time-course analysis of gene expression to identify gene clusters with similar temporal patterns in expression during cellulose fermentation. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a progressively decreasing trend in gene expression. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction, transcription and cellulosomal genes showed an increasing trend in gene expression. Proteomic analysis identified over 50 dockerin- and 6 cohesin-module containing components, including 20 new subunits. The list included several proteins of potential interest that specifically respond to the presence of ‘non-avicel’ substrates in the culture medium. Quantitative proteomic results also highlighted the importance of glycoside hydrolase (GH) family 9 enzymes in crystalline cellulose hydrolysis. Overall, the transcriptomic and proteomic results suggest a well-coordinated temporal and substrate-specific regulation of cellulosomal composition in C. thermocellum.