In recent years, due to the spread of antibiotic resistance, Pseudomonas aeruginosa has emerged as an ESKAPE super-resistant pathogen, posing a major threat to current therapies. Phage therapy is currently one of the most promising treatment methods. In this study, we isolated a novel strongly lytic phage HKPH_J3, which is a linear double-stranded DNA of 38008 bp with a GC content of 64.6 %, and contains no harmful genes. Phage HKPH_J3 is a member of the Casadabanvirus genus, and there are subtle genetic differences between it and homologous phages. Phenotypic analysis revealed that phage HKPH_J3 has efficient and stable lytic activity and strongly inhibits and degrades the biofilm of P. aeruginosa PAO1. Moreover, the phage HKPH_J3 significantly inhibited the cytotoxicity of P. aeruginosa PAO1, and in the G. mellonella model, phage HKPH_J3 significantly improved larval survival. In addition, we studied the phage-resistant P. aeruginosa mutant J3yd_PAO1. The infection pressure of phage HKPH_J3 causes a nonsense mutation in the type IV pili (T4P) biogenic protein PilP of P. aeruginosa PAO1, which hinders the folding of the functional domain of the PilP protein and may affect the expression of type IV pili (T4P), inhibiting the adsorption of phage HKPH_J3 and ultimately leading to phage resistance. In summary, phage HKPH_J3 has practical application value in treating drug-resistant P. aeruginosa infections. However, the development of phage resistance in bacteria hinders their application, and the resistance mechanism of bacteria is a key strategy for their survival and reproduction. And, the potential mechanism of bacteria-phage interaction is still unclear. Therefore, we investigated the phage-resistance mechanism of J3yd_PAO1, which helps to increase our understanding of phage-resistant regulation and lays the foundation for the application of phage therapy and the study of bacteria-phage evolution mechanisms.