The study of the interaction between temperate phages and bacteria is vital to understand their role in the development of human diseases. In this study, a novel temperate phage, vB_EcoP_ZX5, with a genome size of 39,565 bp, was isolated from human fecal samples. It has a short tail and belongs to the genus and the family . Phage vB_EcoP_ZX5 encodes three lysogeny-related proteins (ORF12, ORF21, and ORF4) and can be integrated into the 3'-end of of its host YO1 for stable transmission to offspring bacteria. Phage vB_EcoP_ZX5 in lysogenized YO1+ was induced spontaneously, with a free phage titer of 10 PFU/mL. The integration of vB_EcoP_ZX5 had no significant effect on growth, biofilm, environmental stress response, antibiotic sensitivity, adherence to HeLa cells, and virulence of YO1. The ORF4 anti-repressor, ORF12 integrase, and ORF21 repressors that affect the lytic-lysogenic cycle of vB_EcoP_ZX5 were verified by protein overexpression. We could tell from changes of the number of total phages and the transcription level of phage genes that repressor protein is the key determinant of lytic-to-lysogenic conversion, and anti-repressor protein promotes the conversion from lysogenic cycle to lytic cycle.