Introductory ParagraphLong-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on lysine 40 of α-tubulin (αK40), acetylation takes place after stabilization1 and does not protect against depolymerization2. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules3. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible from MAPs and motors1,4, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damages when subjected to repeated stresses, long-lived microtubules are protected from mechanical aging through their acquisition of αK40 acetylation. Thus, unlike other tubulin post-translational modifications that act through MAPs, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.