The increasing attention given to the potential risk offered by enterohepatic Helicobacter species to the well-being of human beings and animals is of significant importance. Helicobacter mastomyrinus (H. mastomyrinus), a bacterium predominantly associated with rodents, has been implicated in liver and intestinal pathologies. Here, a strain of H. mastomyrinus, designated as Hm-17 (GenBank: CP145316.1), was isolated from asymptomatic C57BL/6 mice. Subsequently, an in-depth and comprehensive investigation was undertaken, which included genome sequencing analysis, micro-biochemical identification, evaluation of growth characteristic, cytotoxicity assessment, and testing of animal pathogenicity. The analysis of 16 S rRNA reveals a close phylogenetic relationship between H. mastomyrinus and H. canadensis. However, core-pan genome analysis and an evaluation of pathogenic factors indicates a more robust association between H. mastomyrinus and H. hepaticus. Cytotoxicity analysis revealed that Hm-17 exhibits robust cytolethal distending toxin (CDT) activity, inducing pronounced cellular swelling and death. Furthermore, Hm-17 infection in BALB/c mice results in rapid and characteristic focal necrotic hepatitis. Genome sequencing and pathogenicity analysis indicate that H. mastomyrinus isolates from asymptomatic mice possess significant pathogenic potential. These findings underscore the need for further investigation into the epidemiology and mechanisms of pathogenesis associated with this organism.