Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.