Complement component C8, which mediates membrane attack complex formation and bacterial lysis, plays important roles in the complement system. The cDNA sequences of the C8α, C8β and C8γ genes were cloned from half-smooth tongue sole (Cynoglossus semilaevis). Full-length cDNA of CsC8α (C8α of C. semilaevis), CsC8β and CsC8γ was 1990, 2219 and 886 bp, respectively, which contained open reading frames of 1797, 1749 and 666 bp, encoding 598, 582 and 221 amino acids, respectively. The deduced proteins of CsC8α, CsC8β and CsC8γ showed the closest amino acid similarity to C8α (73%) of Siniperca chuatsi, C8β (76%) of Oryzias latipes and C8γ (72%) of Takifugu rubripes, respectively. The highest expression level of CsC8α, CsC8β and CsC8γ among the 13 normal tissues was observed in liver tissue, followed by much lower levels in other tissues. After infection with Vibrio anguillarum, CsC8α, CsC8β and CsC8γ were significantly up-regulated in all of the detected tissues, including the intestine, liver, gill, head kidney, blood and spleen. Then, a recombinant expression plasmid was constructed, and the recombinant CsC8α protein was expressed in GS115 pichia pastoris yeast. Furthermore, to investigate the biological functions of recombinant CsC8α, an antibacterial assay was performed, and the results showed that recombinant CsC8α obviously inhibited growth of V. anguillarum, Edwardsiella tarda and Vibrio parahaemolyticus. Taken together, these results suggest that CsC8α, CsC8β and CsC8γ may play important roles in the immune defense of C. semilaevis.