E2F transcription factors control cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, whether nuclear translocation of E2F4 alone is sufficient to trigger intestinal epithelial cell proliferation remains to be established. Adenoviruses expressing fusion proteins between GFP and wild-type (wt)E2F4 or GFP and NLS-tagged E2F4 were used to infect normal human intestinal epithelial crypt cells (HIEC). In contrast to expression of wtE2F4, persistent expression of E2F4 into the nucleus of HIEC triggered phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis, formation of apoptotic bodies and activation of caspase-9 and caspase-3. Inhibition of caspase activities by zVAD-fmk partially inhibited cell death induced by E2F4-NLS. An induction of p53, phosphorylated Ser15-p53, PUMA, FAS, BAX, RIP, phosphorylated JNK1 was also observed in HIEC expressing E2F4-NLS comparatively to wtE2F4-expressing cells. E2F1 and p14ARF expression remained unaltered. Down-regulation of p53 expression by RNA interference attenuated cell death induced by E2F4-NLS. By contrast, the level of cell death was negligible in colon cancer cells despite the strong expression of E2F4 into the nucleus. In conclusion, deregulated nuclear E2F4 expression induces apoptosis via multiple pathways in normal intestinal epithelial cells but not in colon cancer cells. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but at the same time accelerate cell death. However, intestinal cells acquiring mutations (e.g. p53, Bax loci...) may escape apoptosis, thereby revealing the full mitogenic potential of the E2F4 transcription factor.