BACKGROUND: The functional diversity of microtubules is regulated through the expression of distinct α- and β-tubulin isotypes together with several post-translational modifications, a concept known as tubulin code. Tubulin detyrosination is a reversible post-translational modification which consists in the removal of the genetically encoded C-terminal tyrosine residue of most α-tubulins. While this modification has been observed in the megakaryocyte lineage, its importance remains poorly understood in platelet biogenesis. OBJECTIVES: To assess the role of α-tubulin detyrosination in platelet biogenesis. METHODS: The responsible enzymes and the relative abundance of detyrosinated α-tubulins were monitored by qRT-PCR and Western Blotting, respectively, in human cultured megakaryocytes and platelets differentiated from CD34 RESULTS: Transcriptional analysis identified VASH1-SVBP and MATCAP as the predominant detyrosinases in the megakaryocyte lineage. During megakaryocyte maturation, their transcript levels progressively increased and correlated with an accumulation of detyrosinated α-tubulins. Remarkably, inhibition of VASH1-SVBP by EpoY abolished tubulin detyrosination, establishing VASH1-SVBP as the main functional detyrosinase in megakaryocytes. More importantly, EpoY enhanced proplatelet formation and platelet production in vitro. These in vitro data were confirmed in vivo in SVBP-deficient mice, which exhibited an increase in platelet counts. CONCLUSION: These findings reveal, for the first time, a role for tubulin detyrosination in proplatelet formation, thereby expanding our understanding of the megakaryocyte tubulin code beyond tubulin isotypes.