• Availability of next generation sequencing technology has increased the discovery rate of human O -glycosylation disorders. • The vast phenotypic variability of O -glycosylation disorders reflects the large diversity of O -glycan structures. • Studies of O -glycosylation disorders have revealed tissue-specific O -glycosylation pathways and protein targets. • Modeling patient mutations in structural models of O -glycan enzymes facilitates characterization of structure-function relationships. • Advances in O -glycoproteomics are key to unravel the human O -glycosylation machinery. Over 100 human Congenital Disorders of Glycosylation (CDG) have been described. Of these, about 30% reside in the O -glycosylation pathway. O -glycosylation disorders are characterized by a high phenotypic variability, reflecting the large diversity of O -glycan structures. In contrast to N -glycosylation disorders, a generic biochemical screening test is lacking, which limits the identification of novel O -glycosylation disorders. The emergence of next generation sequencing (NGS) and O -glycoproteomics technologies have changed this situation, resulting in significant progress to link disease phenotypes with underlying biochemical mechanisms. Here, we review the current knowledge on O -glycosylation disorders, and discuss the biochemical lessons that we can learn on 1) novel glycosyltransferases and metabolic pathways, 2) tissue-specific O -glycosylation mechanisms, 3) O -glycosylation targets and 4) structure-function relationships. Additionally, we provide an outlook on how genetic disorders, O -glycoproteomics and biochemical methods can be combined to answer fundamental questions regarding O -glycan synthesis, structure and function. [ABSTRACT FROM AUTHOR]