The virulent phage ST32 that infects the Escherichia coli strain ST130 was isolated from a wastewater sample in China and analyzed. Morphological observations showed that phage ST32 belongs to the Myoviridae family, as it has an icosahedral capsid and long contractile tail. Host range analysis showed that it exhibits a broad range of hosts including non-pathogenic and pathogenic E. coli strains. Interestingly, phage ST32 had a much larger burst size when amplified at 20 °C as compared to 30 °C or 37 °C. Its double-stranded DNA genome was sequenced and found to contain 53,092 bp with a GC content of 44.14%. Seventy-nine open reading frames (ORFs) were identified and annotated as well as a tRNA-Arg. Only nineteen ORFs were assigned putative functions. A phylogenetic tree using the large terminase subunit revealed a close relatedness with four unclassified Myoviridae phages. A comparative genomic analysis of these phages showed that the Enterobacteria phage phiEcoM-GJ1 is the closest relative to ST32 and shares the same new branch in the phylogenetic tree. Still, these two phages share only 47 of 79 ORFs with more than 90% identity. Phage ST32 has unique characteristics that make it a potential biological control agent under specific conditions.