TLR25 is a new member of TLR1 family that is only identified in teleosts, but its function in immune response isstill unclear. In current study, the coding sequence (CDS) of TLR25 was cloned from Schizothorax prenanti(named spTLR25), and spTLR25 is 2454 bp in length and coding a protein of 817 aa. The spTLR25 contains asignal peptide, twenty leucine-rich repeat (LRR) domains, a LRR C-terminal (LRRCT) motif, a transmembraneregion and a Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis indicates that spTLR25 has the closestrelationship with Cyprinus carpio (C. carpio) TLR25-2. The 3D structure of spTLR25 exhibits 5 α-helices and 3 β-sheets in the TIR domain, and 8 α-helices and 6 β-sheets in the LRR domains. The spTLR25 is mainly expressed inimmune-related tissues and peripheral blood leukocytes (PBL). Furthermore, the expression levels of spTLR25were upregulated in spleen, head kidney and liver while S. prenanti was challenged with LPS or Aeromonashydrophila (Ah), and the upregulation was also detected in head kidney leukocytes (HKL) after LPS and Poly (I:C)stimulation. The luciferase reporter assay demonstrated that NF-κB and type I IFNs signaling pathways can beactivated by spTLR25, and this process may involve in the cascade amplification of TLR25-MyD88 signaling. Inaddition, the co-localization analysis showed that spTLR25 localizes to intracellular region. Taken together, ourresults reveal that teleost-specific TLR25 may be a multifunctional receptor for recognizing both LPS and Poly(I:C) and may activate NF-κB and type I IFNs signaling pathways.