Since the clinical success of cisplatin, there is a growing demand for cytotoxic metal complexes able to overcome the limitations associated with cisplatin therapy. It has been extensively demonstrated that its cytotoxic activity is due to interaction with DNA. In fact, cisplatin interferes with DNA replication and transcription processes, kinking the DNA duplex by covalently binding two adjacent guanines in the major groove. Unfortunately, the use of cisplatin is associated with undesirable side effects. Therefore, a second generation of Pt-based complexes (oxaliplatin, carboplatin), and a series of non-Pt-based complexes, with different mechanisms of action, have been developed. In this frame, dirhodium tetracarboxylates, especially dirhodium tetraacetate [Rh2(μ-O2CCH3)4], attracted great interest. The paddlewheel dirhodium tetraacetate complex has been considered a potential anticancer compound since it exhibits an appreciable carcinostatic activity. Information on the interaction of this metallodrug with DNA are limited. To obtain insights on its mechanism of action, X-ray crystallography and mass spectrometry have been carried out. The X-ray structure of the dirhodium/DNA adduct reveals a bimetallic center bound to an adenine via axial coordination. This result is significant since dirhodium mode of binding to DNA is different from that of cisplatin, which binds guanines. This view is supported by ESI MS experiments, pointing out that, at short incubation times, adducts are formed between the DNA and [Rh2(μ-O2CCH3)4] in agreement with crystallographic results. Furthermore, for longer incubation times, the dirhodium tetraacetate converts into a mono-rhodium tetraacetate fragment as the consequence of progressive cleavage of the Rh-Rh bond.