Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanism s of F. psychrophilum in causing the disease are poorly understood. Currently t he control of BCWD mainly relies on the use of antibiotics. E fficient and universal vaccines against this disease are not commercially available. One of the major obstacles for the identification of virulence factors and development of live attenuated vaccines in F. psychrophilum is the difficulties in genetic manipulation. Most strains of this fish pathogen are resistant to foreign DNA transfer, due to the presence of the protective restriction-modification (RM) “immune” systems. In this study, we identified two critical methyltransferases, HpaIIM and ScrFIM, belonging to the type II RM systems, in F. psychrophilum CSF259-93. A helper plasmid pSS05 carrying both HpaIIM and ScrFIM encoding genes was constructed, and was shown to protect the target DNA from restriction digestion and ensure successful conjugative DNA transfer from Escherichia coli to F. psychrophilum CSF259-93. By using pSS05 and a previously developed sacB -mediated deletion system, we constructed the first gene deletion mutant in F. psychrophilum CSF259-93. The mutant lacking gldN, a core component of the T9SS, is deficient in secreted proteolytic activity, colony spreading, single cell motility, and virulence on rainbow trout. G enomic analysis of 16 F. psychrophilum strains, isolated in Chile, China, Denmark, Finland, France, and the United States, revealed the strains that are phylogenetically close tend to carry the same RM systems , and the HpaII and ScrFI RM systems are present in 13 of the 16 analyzed genomes. We further found the pre-methylation system developed in this study functions in most of the 16 strains. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines in multiple F. psychrophilum strains to prevent BCWD.