Consumption of grape seed extract (GSE) is widely marketed as a dietary supplement and is considered safe for human health. Nevertheless, the analytical composition of GSE from different grape cultivars, growing in special agronomic constraints, differs greatly in flavan-3-ols content. The major concern with GSE studies is a lack of availability of uniformly standardised preparations, which raises an important question whether different GSE samples have comparable activity and trigger the same mechanisms of action on a given biological system. Therefore, it is tempting to speculate that GSE, obtained from different cultivars, could exert differentiated anticancer effects. The focus of the present study is to determine the selective biological efficacy of GSE obtained from three different sources on the human colon cancer cell line Caco-2. Irrespective of its source, high doses of GSE induced a significant inhibition on Caco-2 cell growth. Moreover, apoptosis was enhanced through both caspase-dependent and caspase-independent mechanisms, leading to an early apoptosis-inducing factor release and, further, to a dramatic increase in caspase 7 and 3 activity. However, a significant difference in apoptotic rates induced by the three grape sources clearly emerged when treating cancer cells with low and intermediate GSE concentrations (25 and 50 μg/ml).