Citrobacter freundii, a Gram-negative bacterium, causes many opportunistic infections. Bacteriophage phiCFP-1 was isolated and characterized by its ability to lyse the multidrug-resistant clinical C. freundii strain P10159. Transmission electron microscopy showed that the phage has an icosahedral head and a short tail, making it a Podoviridae family member. In a single-step growth experiment, phiCFP-1 exhibited an eclipse period of 20 min and a burst size of 100 particles per cell. Its genome assembled as a circular molecule when genomic sequencing was completed. However, based on genome content and organization, it was categorized as a classic T7-related phage, and such phages are known to have linear genomes with direct terminal repeats. With the quick and simple method established herein, the 38,625-bp linear double-stranded DNA with 229-bp direct terminal repeats was accurately identified. The genome contained 43 putative open reading frames and no tRNA genes. Using a proteomics-based approach, seven viral and two host proteins from purified phiCFP-1 particles were identified. Comparative genomics and recombination analyzes revealed close genetic relatedness among phiCFP-1, phiYeO3-12/vB_YenP_AP5 (from Yersinia enterocolitica O3), and phiSG-JL2 (from Salmonella enterica). J. Med. Virol. 88:895–905, 2016. © 2015 Wiley Periodicals, Inc.