Abstract: In recent years, overwintering syndrome has severely impacted the aquaculture of freshwater fish such as grass carp (Ctenopharyngodon idella). To investigate its pathogen and pathogenic mechanism, we isolated and identified the pathogenic bacteria from grass carp with typical overwintering syndrome, followed by analyses of its biological characteristics and whole-genome sequencing. A predominant strain, designated Ci2402, was isolated from the internal organs of diseased grass carp. The strain was identified as Flavobacterium psychrophilum based on 16S rRNA sequencing and genomic phylogenetic analysis, showing the highest genetic relationship to the crucian carp-derived F. psychrophilum strain NJ01. Strain Ci2402 exhibited characteristics including gliding ability, extracellular protease activity, hemolytic activity, and thermolability. This strain belonged to serotype Type 1 and ST51. Antimicrobial susceptibility testing reveals that it was sensitive to enrofloxacin, florfenicol, neomycin, doxycycline, and trimethoprim/sulfamethoxazole. Artificial infection experiments demonstrate that Ci2402 was pathogenic to grass carp under high-dose infection conditions. Whole-genome sequencing analysis reveals that Ci2402 harbored Sec secretion system, T9SS secretion system, as well as virulence-related genes such as collagenase, protease, and hemolysin. To sum up, F. psychrophilum Ci2402 is a pathogenic bacterium causing grass carp overwintering syndrome, exhibiting extracellular protease activity, hemolytic activity, and thermolability, with multiple virulence genes in its genome. This study facilitates the elucidation of overwintering syndrome etiology and pathogenic mechanisms in grass carp.