The effect of daily contact of a grape seed extract (GSE) on Caco-2 cell proliferation and differentiation was investigated. GSE at 400 mg/L was added to Caco-2 cells for 2 h a day after successive incubation in saliva, gastric, and pancreatic media. When applied at the beginning of the cell culture, GSE triggered inhibition of cell growth associated with a possible cytotoxic reaction. On the other hand, when the treatment was applied to confluent cells, treated cells displayed a higher protein content than control cells and a more developed brush border, with taller and denser microvilli. These observations were accompanied by stimulation of alkaline phosphatase activity, especially at day 5 postconfluency, with a 2.2-fold increase in comparison with the control. On the other hand, aminopeptidase N activity was inhibited throughout the differentiation period in GSE-treated cells to reach 28.8% of control cell activity on day 30. GSE did not affect either sucrase-isomaltase activity or cytoplasmic lactate dehydrogenase (LDH) activity, which otherwise appeared to be a good cellular marker. GSE treatment of Caco-2 cells thus inhibited their proliferation from seeding onward and stimulated both proliferation and differentiation after confluency.