The ASPP (apoptosis-stimulating protein of p53) protein family plays very key roles in apoptosis regulation, in both p53-dependent and p53-independent pathways. However, the ASPP homologous gene has not been identified in amphioxus to date. Here, we identified and characterized an ASPP gene from Branchiostoma belcheri (designed as AmphiASPP) and extensively studied its evolution and roles involved in innate immunity. The results showed that the amphioxus genome has an ASPP homolog gene with an ORF of 3285 bp, encoding 1094 amino acids which contains ANK repeats and SK3 domain. The evolutionary analyses indicated that the members of ASPP protein family might be present in a common ancestor of Nematostella vectensis and underwent positive selective in the evolutionary history. In addition, the amphioxus ASPP gene was ubiquitously and differentially expressed in five investigated tissues, and the amphioxus ASPP gene was involved in the innate immune response of LPS and LTA stimulation. Finally, bioinformatic analyses displayed that amphioxus ASPP protein could interact with REL protein by conserved binding sites compared with human ASPP2 protein, which seemed to further suggest that the amphioxus ASPP protein involve in innate immunity through NF-кB signaling pathway. Taken together, our findings provided an insight into the evolution and innate immunity function of the ASPP family.