Alternative splicing can produce a variety of splicing isoforms to increase protein diversity, participate in the regulation of gene expression and the occurrence and development of diseases, and thus play an important role in innate immunity. Ubiquitin like with PHD and ring finger domains 2 (Uhrf2) protein is associated with cell proliferation, inflammation, tumors, and cancer, and is currently the focus of medical immunology research, but there is little research on alternative splicing of the Uhrf2 gene. In this study, we identified two different splicing isoforms of Uhrf2 in Miichthys miiuy through Sanger sequencing, dual-luciferase reporter gene assay, qRT-PCR, subcellular localization experiments, and named them Uhrf2-α and Uhrf2-β. Subcellular localization experiments found that Uhrf2-α was mainly located in the nucleus, while Uhrf2-β was mainly located in the cytoplasm. Although their localization was different, both could significantly inhibit the activation of IRF3 and NF-κB signaling pathways, and effectively inhibit the levels of inflammatory cytokines. These results indicate that Uhrf2-α and Uhrf2-β play important negative regulatory roles in innate immune responses in fish.