Mammalian Nedd4 family interacting protein 1 (Ndfip1) serves as an activator of the E3 ubiquitin ligase, promoting ubiquitination and limiting the production of pro-inflammatory cytokines. However, the functional role of teleost Ndfip1 is not completely understood. In the current study, an Ndfip1 gene designated as OnNdfip1 was characterized in Nile tilapia. Then we investigated the expression pattern, its interaction with HECT E3 ligase OnNEDD4 or OnItch, and the regulation of NF-κB signaling. The expression of OnNdfip1 in several tissues was detected by qPCR. Co-immunoprecipitation and ubiquitination assay were performed to detect the potential mechanisms. Our study revealed that OnNdfip1 exhibited widespread expression across various tissues, with its expression pattern demonstrating significant changes following stimulation with S. agalactiae or poly I:C. Notably, OnNdfip1 was found to interact with OnItch or OnNEDD4. Functionally, overexpression of OnNdfip1 did not promote the ubiquitination of Tak1 and did not influence NF-κB regulation by Itch, while it significantly inhibited NF-κB signaling induced by OnTak1. This study represents the first characterization on the functional role of teleost Ndfip1 and elucidates its involvement in the signal regulation.