The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σ) is employed to modulate gene transcription, and σ is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a mutant (Δ), in which σ is assumed to be "always on," was generated. Δ did not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. Δ was defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand the unique phenotype of Δ showed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σ-controlled regulators, including the pleiotropic regulator sinRR'. While these profiles were distinct to Δ, changes in some σ-controlled stress-associated genes were similar to those reported in the absence of σ. Further analysis of Δ showed unexpected lower intracellular levels of σ, suggesting an additional post-translational control mechanism for σ in the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses in C. difficile. Pathogens like Clostridioides difficile face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σ) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provide C. difficile with the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a mutant in growth, persistence, and virulence and suggest alternate σ control mechanisms in C. difficile. Understanding C. difficile responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.