β-tubulin isotypes exhibit similar sequences but different activities, suggesting that limited sequence divergence is functionally important. We investigated this hypothesis for TUBB3/β3, a β-tubulin linked to aggressive cancers and chemoresistance in humans. We created mutant yeast strains with β-tubulin alleles that mimic variant residues in β3 and find that residues at the lateral interface are sufficient to alter microtubule dynamics and response to microtubule targeting agents. In HeLa cells, β3 overexpression decreases the lifetime of microtubule growth, and this requires residues at the lateral interface. These microtubules exhibit a shorter region of EB binding at the plus end, suggesting faster lattice maturation, and resist stabilization by paclitaxel. Resistance requires the H1-S2 and H2-S3 regions at the lateral interface of β3. Our results identify the mechanistic origins of the unique activity of β3 tubulin and suggest that tubulin isotype expression may tune the rate of lattice maturation at growing microtubule plus ends in cells. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].