Microtubules, the key component of the cytoskeleton, are indispensable for various cellular tasks such as cell differentiation, mitosis, etc. Disruption of microtubule assembly plays a crucial role in tumor regulation. Protein l-isoaspartyl methyltransferase (PIMT), found in abundance in brain, is known to repair abnormal isoaspartate residues, formed on aging, to the normal aspartate. Using various biophysical techniques, we show that PIMT can compromise the microtubule network after internalization in cells. The binding of PIMT to tubulin overlaps with the vinblastine binding site, as inferred from the competitive assay. Experiments using MCF-7 breast cancer cells revealed the binding of PIMT with intracellular tubulin and the disruption of the network. Results are reproducible in an additional breast cancer cell line, MDA-MB-231. In vivo experiments using mice with breast cancer cells revealed tumor regression after treatment with PIMT. Like other antimitotic agents, PIMT can target tubulin, regulating the structure and dynamics of microtubules. The free energy of binding of PIMT to tubulin was found to be -6.3 kcal/mol obtained from Isothermal Titration Calorimetry (ITC). The PIMT-tubulin complex structure determined by AlphaFold suggests an interface that overlays considerably with that between two tubulin heterodimers in the microtubule, suggesting a mechanism by which the binding of PIMT can induce the dissociation of the microtubule. Asp/Asn residues present at the latter interface would be subjected to various degrees of isomerization and thereby control the properties and functions of the microtubule and also be a substrate for the repair enzyme, PIMT.