Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, β-1,3 N-acetylglucosaminyltransferase 1 and β-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes. Synopsis SPPL3 is a highly conserved eukaryotic intramembrane-cleaving GxGD-type aspartyl protease of undefined function. We show that SPPL3 liberates medial/trans-Golgi glycosyltransferases from their N-terminal membrane anchors to regulate the intracellular pool of active Golgi glycosyltransferases and the extent of N-glycan decoration of cellular glycoproteins. Loss of SPPL3 in vitro and in vivo is associated with more extensive N-glycosylation. Overexpression of active SPPL3, but not of an inactive mutant, leads to less extensive N-glycosylation. Constitutive secretion of Golgi glycosyltransferases such as GnT-V, β3GnT1 and β4GalT1 is dependent on cellular SPPL3 activity. SPPL3-dependent GnT-V endoproteolysis occurs close to GnT-V's predicted transmembrane domain. Changes in SPPL3 expression strongly affect intracellular glycosyltransferase levels, explaining the observed alterations in N-glycan composition. The intramembrane-cleaving GxGD-type aspartyl protease, SPPL3, controls the proteolytic release of the ectodomain of glycosyltransferases and glycosidases to regulate cellular N-glycosylation.