Mechanical signals from the tumor microenvironment modulate cell mechanics and influence cell metabolism to promote cancer aggressiveness. Cells withstand external forces by adjusting the stiffness of their cytoskeleton. Microtubules (MTs) act as compression-bearing elements. Yet how cancer cells regulate MT dynamic in response to the locally constrained environment has remained unclear. Using breast cancer as a model of a disease in which mechanical signaling promotes disease progression, we show that matrix stiffening rewires glutamine metabolism to promote MT glutamylation and force MT stabilization, thereby promoting cell invasion. Pharmacologic inhibition of glutamine metabolism decreased MT glutamylation and affected their mechanical stabilization. Similarly, decreased MT glutamylation by overexpressing tubulin mutants lacking glutamylation site(s) decreased MT stability, thereby hampering cancer aggressiveness in vitro and in vivo. Together, our results decipher part of the enigmatic tubulin code that coordinates the fine-tunable properties of MT and link cell metabolism to MT dynamics and cancer aggressiveness. Display omitted •Matrix stiffening stabilizes MT•Mechano-dependent MT stabilization relies on MT glutamylation•MT glutamylation relies on mechano-induced glutamine catabolism•MT glutamylation guides breast tumor progression Torrino et al. identify mechano-activated glutamine catabolism as a necessary metabolic pathway for microtubule (MT) stabilization, connecting, for the first time, the mechanical forces and cell metabolism to MT dynamics. Matrix stiffening promotes glutamine-dependent MT glutamylation and thereby stabilizes MTs. Hampering MT glutamylation blunts breast cancer progression