Bile salts are important components in solubilizing lipid from the diet, which also possess antimicrobial properties by solubilizing bacteria membranes. For Enterobacteriaceae to survive in the gut environment, they develop multiple mechanisms to gain intrinsic resistance, mainly in two ways: restricting the entrance of bile molecules by the asymmetric outer membrane (OM); pumping out them by efflux pumps. The OM with a well-packed low-fluidity lipopolysaccharide layer at outer leaflet restricts toxic compounds from entry, which allows Gram-negative bacteria to live under various harsh conditions. Here we find that the OmpC-Mla system, which has been shown previously to maintain the OM asymmetry by retrogradedly removing PLs from the outer leaflet of the OM back to the inner membrane, has a novel role in conferring cell bile salt resistance. We show that cells have defects in the OmpC-Mla system are sensitive to bile salts, and this sensitivity is independent of the defects in OM asymmetry. Also, we demonstrate bile salts physically interact with MlaD, one of the inner membrane components of the OmpC-Mla system.