CD40 is known as “master switch” in immune response to pathogen infection in mammals. However, limited information of CD40 is known in lower vertebrates. In this study, a novel CD40 homolog (Ls-CD40) was cloned and characterized from humphead snapper, Lutjanus sanguineus. The Ls-CD40 cDNA composed of 2073 bp with a 69 bp of 5′-UTR, a 1020 bp of 3′-UTR and an open reading frame (ORF) of 984 bp, encoding 327 amino acid residues. Sequence analysis showed that Ls-CD40 contained a single peptide, a transmembrane domain and four cysteine-rich domains. The deduced amino acid sequence of Ls-CD40 shared 40%–53% identities with other known fish CD40. The qRT-PCR showed that Ls-CD40 gene expressed in all examined tissues with the most abundant in spleen and lowest level in intestine. After V. harveyi and poly I:C stimulation, the expression of CD40 were significantly induced in spleen. Moreover, Ls-CD40 could interact with Ls-TRAF3 in vitro. These data indicate that Ls-CD40 might play a regulatory role in immune response of L. sanguineus.