[Background] Yersinia enterocolitica, an important food-borne zoonotic pathogen, is commonly exposed to cell envelope stresses due to its living environment and epidemic properties. The envelope stress responses of Y. enterocolitica play a very important role in maintaining its environmental tolerance and virulence. [Objective] Explore the role of Y. enterocolitica in regulation of cell envelope stress. [Methods] Polymyxin B was used to disrupt the cell membrane stability of Y. enterocolitica, and we explore the response of regulator of capsule synthesis (Rcs) system to the envelop stress by polymyxin B from the changes in growth, motility ability, biofilm formation ability and related gene expression of Y. enterocolitica. [Results] The cell membrane pressure caused by polymyxin B inhibited the motility and biofilm formation of Y. enterocolitica, which were restored after blocking the Rcs signaling pathway. The expression of genes regulated by Rcs system, such as flhC, hmsS and hmsT, were analyzed. The results further confirmed the regulation of Rcs system in response to the membrane pressure induced by polymyxin B. After sensing the envelop stress signals, Rcs system could actively regulate gene expression and cause pleiotropic changes to improve the resistance of Y. enterocolitica to polymyxin B. [Conclusion] This study clarified the specific regulatory role of the Rcs system in response to membrane stress caused by polymyxin B, and will deepen the understanding of the environmental response mechanisms of Y. enterocolitica.