Wortmannin (WTN) is a steroid metabolite that inhibits phosphatidylinositol 3-kinase and other signaling pathways. Structurally, the WTN consists of a cyclopentanophenanthrene-like structure with several oxygen-rich moieties which have the potential to interact with deoxyribonucleic acid (DNA) molecules.We aim to evaluate the WTN and calf thymus DNA (ct-DNA) interaction with molecular docking using the AutoDock 4.2 software. UV and fluorescence spectroscopy and viscosity techniques were performed to confirm the analysis.Molecular docking showed that the WTN interacted with ct-DNA via hydrogen bonds at guanine-rich sequences. The number of hydrogen bonds between the WTN and DNA was 1-2 bonds (average 1.2) per WTN molecule. The binding constant was 2 × 10 M. UV spectroscopy showed that the WTN induced a hyperchromic feature without wavelength shifting. The WTN and DNA interaction led to quenching of DNA-emitted fluorescence. The different concentrations of WTN had no effect on DNA viscosity. Taken together, our results demonstrated WTN interacts with DNA in the nonintercalating mode, which is considered as a new mechanism of action.These results suggest that the WTN may exert its biological effects, at least in part, via interaction with DNA.