Bis-dioxopiperazines have been termed catalytic inhibitors of topoisomerase II to distinguish them from poisons that induce DNA double-strand breaks (DSBs), but ICRF-193 acts as a poison in cells with mutated checkpoint mechanisms. We previously reported that 20-O-ingenolEZ, a catalytic inhibitor, inhibits the ATPase activity of topoisomerase IIa and induces G2 arrest in mouse mammary tumor (MMT) cells. In this study, we investigated the effects of 20-O-ingenolEZ on cells with mutations in the RecQ helicase genes (BLM-/- and WRN-/- DT40 cells) and wild-type DT40 cells. 20-O-ingenolEZ completely inhibited the proliferation of BLM-/- cells, induced phosphorylation of H2AX (a marker of topoisomerase II-mediated DSBs), and the induction of apoptosis in BLM-/- cells showed characteristics of a topoisomerase II poison. It did not inhibit the proliferation of WRN-/- cells. The inhibition of BLM-/- DT40 cell proliferation by 20-O-ingenolEZ was more sensitive than that of wild-type cells. Thus, 20-O-ingenolEZ appears to be a potential anti-proliferative agent against specific cell types, and further studies may provide insights into DNA damage response pathways involving BLM and the development of anti-cancer agents.