Microtubules are a significant therapeutic target for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule stabilizing agent that synergizes with taxoid drugs through a unique site, and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A, and present a structural model of the peloruside binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry (HDX-MS) of different microtubule stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the α/β-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of β-tubulin at a previously unknown ligand site, rather than on α-tubulin as suggested in earlier studies.