Two novel tumor necrosis factor receptors, Bbt-TNFR1 and Bbt-TNFR2, were isolated from Chinese amphioxus, the closest relative to vertebrate. The mRNA of Bbt-TNFR1 encoded a type I membrane protein of 452 amino acids, including four cysteine-rich domains in the extracellular region and a putative TRAF6-binding site at its 154aa long cytoplasmic tail. Bbt-TNFR2 was a 304aa long type I membrane protein, featuring three cysteine-rich domains and a short cytoplasmic tail of just 13 amino acids. Southern blot revealed that Bbt-TNFR1 was a single copy gene, while Bbt-TNFR2 was presented in multiple copies. Sequence comparison indicated that both Bbt-TNFR1 and Bbt-TNFR2 were weakly similar to LT-bR, HVEM, TNFR2, CD40, OX40 and DcR3. Real-time PCR showed that Bbt-TNFR1 and Bbt-TNFR2 were regulated during development and finally had high expression in mucosa-rich tissues in adult stage. Furthermore, up-regulated expression of both genes was also observed in guts after Gram-positive bacteria challenge. However, not like Bbt-TNFR2's slowly and gradually augmentation in the following 48 h, expression of Bbt-TNFR1 dramatically surged up within 4 h and then subsided rapidly. Taking together, Bbt-TNFR1 and Bbt-TNFR2 may involve in the host defense of Chinese amphioxus via distinct fashions.