Complement factor I (CFI) is a novel regulatory serine protease that plays an important role in resistance to pathogen infection. In this study, the CFI gene of Pelteobagrus vachellii (Pv-CFI) was sequenced and characterized. The full-length cDNA of 2320 bp includes a 155 bp 5′-untranslated region (UTR), a 164 bp 3′-UTR, and a 2001 bp open reading frame (ORF) encoding a 667 amino acids. Multiple sequence alignment revealed five highly conserved domains with a typical modular architecture and identical active sites in vertebrates, indicating a conserved function. Pv-CFI mRNA was constitutively expressed in all examined tissues and most abundant in liver. During infection with Aeromonas hydrophila, Pv-CFI mRNA expression was significantly up-regulated in liver at 3–24 h, spleen at 3–48 h and head kidney at 3–48 h. The results suggest Pv-CFI plays an important role in resistance to pathogenic bacteria in P. vachellii.