Abstract Liang, Y.J. and Zhang, S.C. 2006. Demonstration of plasminogen-like protein in amphioxus with implications for the origin of vertebrate liver. —Acta Zoologica (Stockholm) 87: 141–145 Plasminogen, the proenzyme of serine protease plasmin, is a plasma glycoprotein synthesized primarily in the liver, and its evolutionary origin in chordates remains unclear. We demonstrated here that the humoral fluid in amphioxus is capable of cross-reacting with anti-human or anti-mouse plasminogen antibodies, and the hepatic diverticulum in amphioxus is the site of plasminogen-like protein synthesis. The presence of plasminogen-like protein in amphioxus pushes the origin of plasminogen to before the last common ancestor of vertebrates. In addition, the localization of plasminogen-like protein in the hepatic diverticulum suggests that the diverticulum in amphioxus is functionally homologous to the vertebrate liver in respect of plasminogen synthesis, supporting the hypothesis that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.