Recent studies have revealed that LuxS/AI-2 quorum sensing (QS) is the most universal cell-to-cell communication in rumen bacteria; however, it remains unknown how they respond to nutritional stress from a diet shift. This study aimed to explore whether a diet shift could trigger rumen bacterial LuxS/AI-2 QS and its influences on rumen fermentation characteristics and bacterial community diversity and composition. A total of fifteen Hu sheep were selected to undergo a pre-shift diet (Pre, concentrate to forage ratio 75:25) for one month and then abruptly switch to a post-shift diet (Post, concentrate to forage ratio 49:51). Results showed that the serum cortisol and immunoglobulin G concentrations were higher in Post than in Pre (p < 0.05). The microbial density, AI-2 concentration, biofilm formation, and the gene expression of ftsH were higher in Post when compared with Pre (p < 0.05), whilst the gene expression of luxS tended to be lower in Post (p = 0.054). The molar concentration of valerate and fermentation efficiency decreased after the diet shift, while the acetate to propionate ratio and the molar proportion of butyrate were higher in Post compared to Pre (p < 0.05). Moreover, the diet shift increased the richness of ruminal bacteria and the relative abundances of Roseburia, Prevotellaceae UCG-001, and Lachnospira, and decreased the relative abundances of Prevotella, Megasphaera, and Dialister (p < 0.05). A difference in trends was also observed in an analysis of similarity (R = 0.1208 and p = 0.064). This study suggests that a diet shift could trigger rumen bacterial LuxS/AI-2 QS by altering microbial density, AI-2 concentration, biofilm formation, and related gene expression, as well as affect the rumen fermentation pattern and bacterial community diversity and composition. This study may provide insight into a potential strategy for relieving nutritional stress via regulating bacterial communication.