A perfusion culture apparatus, capable of culturing cells having a multilayer structure, includes (i) a film, which is a sheet-shaped carrier on which cells are seeded in a state in which a liquid medium is allowed to pass through the sheet-shaped carrier; (ii) a vessel, which holds the film in a state in which the liquid medium is in contact with front and rear surfaces of the film; and (iii) a gas chamber, which applies pressure from outside to the liquid medium on the side in contact with either of the front and rear surfaces of the film to form a pressure difference between the liquid medium on the side in contact with the one surface and the liquid medium on the side in contact with the other surface, the liquid medium passing through the film in accordance with the pressure difference.