Recently, lytic bacteriophages (phages) have been focused on treating bacterial infectious diseases. We investigated the protective efficacy of a novel Pseudomonas aeruginosa phage, PA1${O}$, in normal and neutropenic mice. A lethal dose of P. aeruginosa PAO1 was administered via the intraperitoneal route and a single dose of PA1${O}$ with different multiplicities of infection (MOI) was treated into infected mice. Immunocompetent mice infected with P. aeruginosa PAO1 were successfully protected by PA1${O}$ of 1 MOI, 10 MOI or 100 MOI with 80% to 100% survival rate. No viable bacteria were found in organ samples after 48 h of the phage treatment. Phage clearing patterns were different in the presence or absence of host bacteria but PA1${O}$ disappeared from all organs after 72 h except spleen in the presence of host bacteria. On the contrary, PA1${O}$ treatment could not protect neutropenic mice infected with P. aeruginosa PAO1 even though could extend their lives for a short time. In in vitro phage-neutrophil bactericidal test, a stronger bactericidal effect was observed in phage-neutrophil co-treatment than in phage single treatment without neutrophils, suggesting phage-neutrophil co-work is essential for the efficient killing of bacteria in the mouse model. In conclusion, PA1${O}$ can be possibly utilized in future phage therapy endeavors since it exhibited strong protective effects against virulent P. aeruginosa infection.