In vitro experiments showed that cell movements are motivated by active actin nucleation and branching at the motile cell’s leading edge. Cortactin and Arp2/3 complex play important roles in this process. Here, we report the expression pattern of these proteins in the gastrula of Amphioxus (Branchiostoma belcheri tsingtaoense) with the method of whole-mount antibody staining; which was indicated that cortactin and Arp2/3 complex were colocalized and mediated the invagination movement during amphioxus gastrulation; and that they appeared in mesendoderm cells but not in ectoderm cells. In addition, a specific stained area near the vegetable pole was detected prior to the initiation of gastrulation.