Several Francisella spp. including F. noatunensis are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella Pathogenicity Island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. PdpA is a member of the pathogenicity determining protein genes encoded by the FPI that are implicated in the ability of the mammalian pathogen, F. tularensis, to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis. Since polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (FnoDpdpA1 and DpdpA2). Both mutants were attenuated (p<0.05), all of which could be restored to wild-type (WT) levels by complementation. Importantly, differences were found for bacterial burden and induction of acute phase and pro-inflammatory genes for Fno-pdpA1 and pdpA2 compared to WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with Fno pdpA1 led to protection (p<0.012) against an acute lethal-dose 40 challenge with WT Fno in the zebrafish model of infection. The results from this study further demonstrate physiological similarities within the genus Francisella relative to their physiological relationships and the utility of zebrafish for addressing virulence factors for the genus.