Phage PH357, a novel lytic Pseudoalteromonas lipolytica phage belonging to the Myoviridae family was isolated from the Yangtze River estuary. The microbiological characterization demonstrated that phage PH357 is stable from -20 to 60 °C and the optimal pH 7. The one-step growth curve showed a latent period of 20 min, a rise period of 20 min, and the average burst size was about 85 virions per cell. Complete genome of phage PH357 was determined. Genome of phage PH357 consisted of a linear, double-stranded 136,203 bp DNA molecule with 34.58% G + C content, and 242 putative open reading frames (ORFs) without tRNA. All the predicted ORFs were classified into eight functional groups, including DNA replication, regulation and nucleotide metabolism, transcription, translation, phage packaging, phage structure, lysis, host or phage interactions, and hypothetical protein. A phylogenetic analysis showed that phage PH357 had similarity to the previously published Pseudoalteromonas phage PH101 and Vibrio phages. Furthermore, the study of phage PH357 genome will provide useful information for further research on the interaction between phages and their hosts.