Exploring the metabolic regulatory mechanisms of bacteria for ramie degumming and constructing more efficient engineered strains are preferred strategies to solve the technical bottleneck of high residual gum content in fibers. Bacillus subtilis strain 168, an advantageous bacterium for microbial degumming, was previously found to significantly up-regulate the expression of bast two-component system (TCS) response regulators CitT, YvcP, and YycI when using ramie as the sole carbon source. In this study, the genes encoding CitT, YvcP, and YycI proteins were knocked out and compared the effects between these gene knockouts and the original strain on the degumming efficiency. The aim was to identify the key TCS response regulators that significantly affect degumming efficiency and to explore the functions of these different response regulators. The results demonstrated that knockout of citT, yvcP, or yycI genes significantly reduced degumming efficiency. Specifically, CitT protein primarily regulated the degradation of pectin, YvcP protein mainly regulated the degradation of hemicellulose, and YycI protein was involved in the regulation of both pectin and hemicellulose degradation. Notably, the absence of CitT protein caused the most significant reduction in degumming efficiency. These findings provide valuable insights into the construction of engineered strains with high degumming efficiency for ramie fibers.