Microtubules (MTs) are key regulators of cellular biology and thus represent potentially critical targets for viral subversion of host-cell function. While MT targeting and modification by viral proteins is frequently observed, the functional outcomes during infection and disease are largely unresolved. Using single molecule localization microscopy, viral reverse genetics, animal infection, immune signalling assays and protein-MT association analysis in live-cells, we perform quantitative sub-diffraction analysis of the outcomes of MT targeting by the rabies virus (RABV) interferon (IFN)-antagonist protein P3. We report that MT-interaction by P3 induces significant bundling of MT filaments, and, by comparing pathogenic and apathogenic strain-derived P3, the capacity to induce bundling correlates with viral pathogencity in vivo. Importantly, we also identify a single mutation of P3 that strongly inhibits MT interaction and bundling and, using genetic manipulation of RABV, we further show that this mutation significantly attenuates virus in vivo. These data implicate P3-induced MT bundling as a novel mechanism of viral suppression of immune signalling and provides new insights into fundamental subcellular processes at the virus-host interface important to lethal infectious disease. These data identify new potential targets for the development of vaccines and antivirals against lyssaviruses, the cause of over 70,000 human deaths world-wide each year.