The present invention provides a method for cultivation of hepatocytes efficiently by using a hepatocyte growth promoting factor derived from 3T3 cells, which comprises culturing hepatocytes isolated from the liver of a matured mammal in a medium containing pleiotrophin, fetal bovine serum, ascorbic acid or analogues thereof and nicotinamide or an analogue thereof, thereby forming primary colonies of the hepatocytes; and a method for subculture of the primary hepatocytes in the same medium.