ABSTRACTThe spread of multidrug resistant and hypervirulentKlebsiella pneumoniaehas recently increased. Phages have been considered alternatives for treating infections caused by tenacious pathogens. Our study describes a novel lyticKlebsiellaphage, hvKpP3, and we obtained spontaneous mutants, hvKpP3R and hvKpP3R15, of hvKpLS8 strain that showing strong resistance to the lytic phage hvKpP3. Sequencing analysis showed that nucleotide-deletion mutations of the glycosyltransferase gene (GT) andwcaJgenes, located in the lipopolysaccharide (LPS) gene cluster and the capsular polysaccharide (CPS) gene cluster, respectively, led to phage resistance. ThewcaJmutation confers the inhibition of phage adsorption by affecting the synthesis of hvKpP3R15 capsular polysaccharide, indicating that the capsule is the main adsorption receptor for bacteriophage hvKpP3. Interestingly, the phage-resistant mutant hvKpP3R has a loss-of-function mutation inGT,which is responsible for lipopolysaccharide biosynthesis. This results in the loss of high-molecular weight lipopolysaccharide (HMW-LPS), and alteration of the lipopolysaccharide structure of the bacterial cell wall confers resistance to phages. In conclusion, our study provides a detailed description of phage hvKpP3 and provides new insights into phage resistance inK. pneumoniae.IMPORTANCEMultidrug-resistant (MDR)Klebsiella pneumoniaestrains pose a particular threat to human health. Therefore, it is very important for us to isolate phage and overcome phage resistance. In this study, we isolated a novel phage belonging to theMyoviridaefamily, hvKpP3, that exhibited high lytic activity against K2 hypervirulentK. pneumoniae. We demonstrated the excellent stability of phage hvKpP3 throughin vitroandin vivoexperiments, indicating its potential as a candidate for future clinical phage therapy. Furthermore, we identified that loss of function in the glycotransferase gene (GT) caused the failure of HMW-LPS synthesis, leading to phage resistance, which provides new insights into phage resistance inK. pneumoniae.