The ubiquitious enzyme topoisomerase I can be targeted by drugs which turn these enzymes into cellular poisons and subsequently induce cell death. Drugs like staurosporine, which do not target topoisomerase I directly, can also lead to stabilization of topoisomerase I-DNA cleavable complexes by an indirect process of reactive oxygen species (ROS) generation and subsequent oxidative DNA damage. In this study, we show that betulinic acid, a catalytic inhibitor of topoisomerases, inhibits the formation of apoptotic topoisomerase I-DNA cleavable complexes in prostate cancer cells induced by drugs like camptothecin, staurosporine, and etoposide. Although events like ROS generation, oxidative DNA damage, and DNA fragmentation were observed after betulinic acid treatment, there is no topoisomerase I-DNA cleavable complex formation, which is a key step in ROS-induced apoptotic processes. We have shown that betulinic acid interacts with cellular topoisomerase I and prohibits its interaction with the oxidatively damaged DNA. Using oligonucleotide containing 8-oxoguanosine modification, we have shown that betulinic acid inhibits its cleavage by topoisomerase I in vitro. Whereas silencing of topoisomerase I gene by small interfering RNA reduces cell death in the case of staurosporine and camptothecin, it cannot substantially reduce betulinic acid-induced cell death. Thus, our study provides evidence that betulinic acid inhibits formation of apoptotic topoisomerase I-DNA complexes and prevents the cellular topoisomerase I from directly participating in the apoptotic process.