Previous studies have shown the existence of a complement system in the amphioxus Branchiostoma japonicum. However, whether it has an opsonic activity similar to that of vertebrates remains unknown. We demonstrated that the humoral fluid (HF) of amphioxus promoted the phagocytosis of yeast cells with sea bass (Lateolabrax japonicus) macrophages, whereas the C3-depleted and heated HF significantly lost the phagocytosis-promoting capacity. In addition, the precipitation of factor B (Bf) led to a marked loss of opsonic activity. Moreover, C3 fragments in the HF were found to bind to yeast cell surfaces. The results indicate that the amphioxus complement system is an important element involved in the opsonic activity, which promotes the sea bass macrophage phagocytosis by tagging yeast cells with C3 fragments via the activation of alternative complement pathway.