Feline calicivirus (FCV) is a key pathogen causing upper respiratory diseases and oral ulcers in cats, with significant genetic variability. This study aimed to isolate the FCV-BJ616 strain, investigate its pathogenic mechanisms, and provide insights for developing antibody therapies and broad-spectrum vaccines. The virus was purified through three rounds of plaque cloning, and its morphology was observed via electron microscopy. Immunofluorescence and Western blotting confirmed VP1 protein expression. Using systems biology and reverse genetics, an infectious clone of rFCV-BJ616 was constructed and recovered. The recombinant virus maintained genetic stability similar to the parental strain. Animal experiments showed that rFCV-BJ616 exhibited wild-type virulence, causing persistent high fever, weight loss, and multi-organ pathology in infected cats. Proteomic analysis revealed that FCV activates cytokine pathways, causing elevatedIL-8 and IFN-βlevels, leading to a cytokine storm. This study enhances understanding of FCV-BJ616's genetic evolution and successfully established a reverse genetics platform for FCV, enabling precise manipulation of the virus at the genomic level and providing a controllable and reproducible experimental basis for systematic studies on its replication, pathogenesis, and vaccine attenuation mechanisms.