Tubulin detyrosination has been implicated in various human disorders and is important for regulating microtubule dynamics. While in most organisms this modification is restricted to α-tubulin, in trypanosomatid parasites, it occurs on both α- and β-tubulin. Here, we show that in , a single vasohibin (LmVASH) enzyme is responsible for differential kinetics of α- and β-tubulin detyrosination. LmVASH knockout parasites, which are completely devoid of detyrosination, show decreased levels of glutamylation and exhibit a strongly diminished pathogenicity in mice, correlating with decreased proliferation in macrophages. Reduced virulence is associated with altered morphogenesis and flagellum remodeling in detyrosination-deficient amastigotes. Flagellum shortening in the absence of detyrosination is caused by hyperactivity of a microtubule-depolymerizing Kinesin-13 homolog, demonstrating its function as a key reader of the trypanosomatid-tubulin code. Taken together, our work establishes the importance of tubulin detyrosination in remodeling the microtubule-based cytoskeleton required for efficient proliferation in the mammalian host. This highlights tubulin detyrosination as a potential target for therapeutic action against leishmaniasis.