Prolonged exposure to estrogen increases breast cancer risk. Estrogen is known to induce chromosomal aberrations, yet the mechanisms by which estrogen promotes genomic instability are not fully understood. Here, we show that exposure of MCF-7 cells to 17β-estradiol (E2) induces DNA double-strand breaks (DSBs), as determined by the formation of γH2AX foci. Foci formation was dependent upon estrogen receptor-α (ERα) and the catalytic activity of the type II topoisomerase, topoisomerase IIβ (topoIIβ). Moreover, we show by chromatin immunoprecipitation that topoIIβ-dependent E2-induced γH2AX localizes to the promoter of the estrogen-inducible gene, trefoil factor 1. E2-induced foci were associated with cyclin A expression and inhibited by pre-incubation with the DNA polymerase inhibitor aphidicolin suggesting that E2-induced DSBs are mediated by progression through S phase. Furthermore, E2-induced γH2AX foci colocalized with Rad51, suggesting that E2-induced DSBs are repaired by homologous recombination. We propose that DNA DSBs formed by the strand-cleaving activity of the topoIIβ-DNA cleavage complex at estrogen-inducible genes can present a barrier to DNA replication, leading to persistent DNA DSBs in ERα-positive breast cancer cells.