BACKGROUND: Lignocellulose is the most abundant renewable bioresource on earth, and its biodegradation and utilization would contribute to the sustainable development of the global environment. Ruminiclostridium papyrosolvens, an anaerobic, mesophilic, and cellulolytic bacterium, produces an enzymatic complex known as the cellulosome. As one of the most highly evolved species among Ruminiclostridium-type species, R. papyrosolvens is particularly relevant for understanding how cellulolytic clostridia modulate their biomass degradation mechanisms in response to diverse carbon sources. RESULTS: Our study investigates the transcriptional responses of Ruminiclostridium papyrosolvens to different carbon sources to understand its lignocellulose utilization. Using RNA-seq, we analyzed gene expression under glucose, cellobiose, xylan, cellulose, and corn stover, identifying distinct metabolic preferences and regulatory responses. We found significant gene expression changes under corn stover compared to other carbon sources, with enrichment in ABC transporters and cell growth pathways. CAZyme gene expression was regulated by TCSs, affecting sugar transporter systems. Metabolic profiling showed R. papyrosolvens produced more complex metabolites during corn stover fermentation, revealing its adaptability to various carbon sources and implications for metabolic engineering. CONCLUSION: This study not only uncovers the intricate response mechanisms of R. papyrosolvens to lignocellulose and its hydrolysates, but it also outlines the strategy for using R. papyrosolvens as a cellulolytic chassis in genetic engineering.