Therapeutic agents that target tubulin and disrupt microtubule dynamics have shown established clinical efficacy against diverse hematological malignancies and solid tumors for decades. However, the structure-activity relationship between therapeutic drugs and prognosis-associated variations in tubulin primary sequences remains unknown. Here, we reveal that tubulin protein families (i.e., isotypes) determine the intrinsic affinities for paclitaxel, a cornerstone chemotherapeutic. Biophysical characterizations and site-directed mutagenesis identify a point mutation, which is distal from the drug-binding pocket, can invert the hypo-sensitivity of human β3-tubulin to paclitaxel. High-resolution (~2.3 Å) cryo-electron microscopy reveals this mutation allosterically remodels the paclitaxel-binding pocket and concurrently enhances the microtubule intrinsic lattice stability. The allosteric network initiated by this paclitaxel-sensitizing mutation extends to α-tubulin E254, reducing its catalytic efficiency for GTP hydrolysis during microtubule assembly. This results in an expanded GTP-cap at growing ends, consistent with the observed preference of EB proteins for dynamic mutant microtubules, and culminates in a significantly lower catastrophe frequency. Furthermore, site-specific genomic integration of this point mutation within the endogenous β3-tubulin locus renders cancer cells profoundly susceptible to paclitaxel. Our findings illuminate how tubulin primary sequences confer divers conformational landscapes that determines the efficacy of paclitaxel, provide a structural and mechanistic blueprint for exploiting tubulin allosteric effect as an approach to design novel microtubule target agent based therapies to circumvent clinical resistance.