To understand phage infection and host cell lysis mechanisms in pathogenic Salmonella, a novel Salmonella enterica serovar Typhimurium-targeting bacteriophage, SPN9CC, belonging to the Podoviridae family was isolated and characterized. The phage infects S. Typhimurium via the O antigen of lipopolysaccharide (LPS) and forms clear plaques with cloudy centers due to lysogen formation. Phylogenetic analysis of phage major capsid proteins revealed that this phage is a member of the lysogen-forming P22-like phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions and host cell lysis gene clusters show very low levels of identity, suggesting that lysogen formation and host cell lysis mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in S. Typhimurium and Escherichia coli hosts revealed that collaboration of these lysis proteins is important for the lysis of both hosts and that holin is a key protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a ΔcI mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge assays revealed that SPN9CCM has shorter eclipse/latency periods and a larger burst size, as well as higher host cell lysis activity, than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol agents similar to virulent phages.