Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of alpha-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of alpha-tubulin, and purified vasohibins removed the C-terminal tyrosine of alpha-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.