The humoral immune responses of amphioxus Branchiostoma belcheri to microbial challenge remain open to date. Here we examined the changes in PO activity in the humoral fluids in amphioxus before and after challenge with Escherichia coli and Vibrio alginolyticus. It was found that PO activity in the humoral fluids is markedly increased by challenge with E. coli and V. alginolyticus; and the microbial challenge results in a significant rise in subunit 2 of the three PO subunits, making PO subunit 2 a marker enzyme responsive to microbial challenge. This is the first report on microbial induction of the immune-related molecules like PO in B. belcheri.