Although E2F1-mediated DNA double-stranded breaks (DSBs) and tetraploid have been extensively studied, the role of E2F1 in mitotic catastrophe is still unknown. We have previously shown that 8-chloro-adenosine (8-Cl-Ado) induces DNA DSBs and aberrant mitosis in human lung cancer cells, followed by delayed apoptosis. Here, we demonstrate that E2F1-mediated DNA damage is implicated in 8-Cl-Ado-induced chromosome missegregation and apoptosis in lung cancer H1299 cells. We showed that E2F1 was accumulated upon 8-Cl-Ado-induced DNA DSBs. Induction of E2F1 by 8-Cl-Ado caused DNA damage in cycling cells including M cells. In contrast, silencing of E2F1 expression decreased 8-Cl-Ado-induced DNA DSBs, particularly eliminated E2F1-mediated mitotic DNA damage. Over-expression of E2F1 and/or 8-Cl-Ado exposure resulted in aberrant mitotic spindles and chromosome segregation errors. Furthermore, over-expression of E2F1 expression enhanced 8-Cl-Ado-induced apoptosis. Together, our data indicate that E2F1-mediated DNA damage, in particular mitotic DNA damage, is an important fraction of 8-Cl-Ado-induced DNA damage, which is implicated in 8-Cl-Ado-induced mitotic catastrophe and delayed apoptosis. Induction of E2F1 by 8-Cl-Ado may contribute at least partly to the drug-inhibited proliferation of cancer cells.