Prunes are the dried fruits of certain cultivars of Prunus domestica L., and are recognized as a health food. The separated ethanol fraction from concentrated prune juice by DIAION HP-20 (PE) was investigated for cytotoxic effects on two different cancer cell lines in vitro. PE dose-dependently reduced the viable cell number of Caco-2, KATO III, but does not reduce the viable cell number of human normal colon fibroblast cells (CCD-18Co) used as a normal cell model. PE treatment for 24 h led to apoptotic changes in Caco-2 such as cell shrinkage and blebbed surfaces due to the convolutions of nuclear and plasma membranes and chromatin condensation, but this was not observed in CCD-18Co. PE induced nucleosomal DNA fragmentation typical of apoptosis in Caco-2 after 24 h of treatment. These results show that PE induced apoptosis in Caco-2. Furthermore, by Caco-2 treatment with H2O2 chelator catalase and Ca2+-chelator BAPTA/AM, the PE-induced cytotoxic pathway was completely blocked, and the viable cell number of Caco-2 was not affected.