The tripartite motif-containing (TRIM) proteins are involved in a wide range of cellular processes, and the role of TRIM1 in immunity has been explored. However, fundamental studies on fish TRIM1 are lacking. In this study, we cloned and characterized TRIM1 cDNA from the Korean rose bitterling, Rhodeus uyekii (RuTRIM1). Two RuTRIM1 isoforms (RuTRIM1-X1 and RuTRIM1-X2) were identified. The coding sequence (CDS) of RuTRIM1-X1 comprised 2157 bp encoding a 718-aa protein, and the CDS of RuTRIM1-X2 comprised 1929 bp encoding a 642-aa protein. Both RuTRIM1 isoforms contained a RING finger domain, B-box 1, B-box 2, coiled-coil domain, COS box, FN3 motif, and PRY/SPRY domain. The deduced RuTRIM1-X1 and RuTRIM1-X2 proteins had high amino acid identity (76.27–98.89%) with orthologs from various other species, and a phylogenetic tree was constructed. RuTRIM1-X1 and RuTRIM1-X2 mRNA were expressed in all tissues examined, with the highest expression levels detected in the hepatopancreas. During early development, RuTRIM1-X1 and RuTRIM1-X2 mRNA levels changed differently from the gastrula period to the first feeding stage. An in vivo ubiquitination assay showed that RuTRIM1 exhibited RING-dependent E3 ubiquitin ligase activity, mainly by comparing RuTRIM1-X2 to RuTRIM1-X1. The subcellular localization of the two RuTRIM1 protein isoforms was characterized, revealing that they formed aggregates in cytoplasmic bodies in Raw264.7cells. Interferon-γ/lipopolysaccharide-induced nuclear factor-κB signaling was negatively regulated by RuTRIM1-X1 and RuTRIM1-X2, and the negative effect was reversed in RING deletion mutants. To our knowledge, this is the first study to characterize fish TRIM1, which may play a role in the inflammatory response.