Microtubule acetylation of lysine 40 of α-tubulin is a hallmark of stable microtubules. This luminal modification is catalyzed by α-tubulin acetyltransferase 1 (αTAT1) and reversed by histone deacetylase 6 (HDAC6). However, acetylation regulation within the microtubule lumen and the influence of lattice architecture on enzymatic activity remain poorly understood. Here, we reconstitute microtubule acetylation in vitro using purified αTAT1 and HDAC6 on microtubules assembled with defined lattice conformations. We show that αTAT1 outweighs HDAC6 enzymatic activity, but its acetylation efficiency decreases upon microtubule damage. Importantly, αTAT1 efficiently acetylates microtubules with expanded lattices, while compacted lattices impede its activity. Our findings reveal that both microtubule integrity and lattice conformation are critical regulators for αTAT1 enzymatic activity, suggesting that dynamic transitions between compacted/expanded and intact/damaged lattices modulate the acetylation pattern of microtubules in cells.