Glycosylation, or the addition of sugars to proteins, is a highly conserved protein modification defined by both the monosaccharide initially added as well as the amino acid to which it is attached. O-Linked glycosylation represents a diverse group of protein modifications occurring on the hydroxyl groups of serine and/or threonine residues. O-Glycosylation can have wide-ranging effects on protein stability and function, which translate into crucial consequences at the organismal level. This review will summarize structural and biological insights into the major O-glycans formed within the secretory apparatus (O-GalNAc, O-Man, O-Fuc, O-Glc and extracellular O-GlcNAc) from studies in the fruit fly Drosophila melanogaster. Drosophila has many advantages for investigating these complex modifications, boasting reduced functional redundancy within gene families, reduced length/complexity of glycan chains and sophisticated genetic tools. Gaining an understanding of the normal cellular and developmental roles of these conserved modifications in Drosophila will provide insight into how changes in O-glycans are involved in human disease and disease susceptibilities.