Plague caused byYersinia pestisremains a public health threat worldwide. Because multidrug-resistantY. pestisstrains have been found in both humans and animals, phage therapy has attracted increasing attention as an alternative strategy against plague. However, phage resistance is a potential drawback of phage therapies, and the mechanism of phage resistance inY. pestisis yet to be investigated. In this study, we obtained a bacteriophage-resistant strain ofY. pestis(S56) by continuously challengingY. pestis614F with the bacteriophage Yep-phi. Genome analysis identified three mutations in strain S56:waaA* (9-bp in-frame deletion249GTCATCGTG257),cmk*(10-bp frameshift deletion15CCGGTGATAA24), andail*(1-bp frameshift deletion A538). WaaA (3-deoxy-D-manno-octulosonic acid transferase) is a key enzyme in lipopolysaccharide biosynthesis. ThewaaA*mutation leads to decreased phage adsorption because of the failure to synthesize the lipopolysaccharide core. The mutation incmk(encoding cytidine monophosphate kinase) increased phage resistance, independent of phage adsorption, and causedin vitrogrowth defects inY. pestis.The mutation inailinhibited phage adsorption while restoring the growth of thewaaAnull mutant and accelerating the growth of thecmknull mutant. Our results confirmed that mutations in the WaaA–Cmk–Ail cascade inY. pestiscontribute to resistance against bacteriophage. Our findings help in understanding the interactions betweenY. pestisand its phages.