Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) widely expressed on the cell surface and within the extracellular matrix. Composed of alternating β1,4-N-acetylgalactosamine (GalNAc) and β1,3-glucuronic acid (GlcA) disaccharide units, CS is covalently linked to proteoglycan (PG) core proteins via a conserved tetrasaccharide linkage region. Functionally, CS contributes to membrane flexibility, mechanical strength, tissue homeostasis, and cell signaling. Clinically, CS supplementation has demonstrated therapeutic efficacy in osteoarthritis, inflammation, and cataracts, and CS dysregulation has been implicated in neurodegenerative diseases including Parkinson’s, Alzheimer’s, and multiple sclerosis. There are four enzymes involved in CS polymer elongation- CHPF1, CHPF2, CHSY1, and CHSY3- whose specific activities, consensus nomenclature, and overarching interactions remain incompletely understood. These enzymes also belong to a diverse family of glycosyltransferases (GTs), which catalyze glycosidic bond formation across all domains of life. Notably, CS synthases incorporate two GT-A fold domains, reflecting both their evolutionary divergence and substrate specificity. Understanding the domain architecture and functional specialization of GTs involved in CS elongation is crucial for unraveling the mechanistic nuances of GAG biosynthesis and its broader implications in health and disease. In this dissertation, we address these gaps by exploring the structure-function relationships and mechanisms to contribute new biochemical and structural insights into the GTs that synthesize chondroitin sulfate. Chapter 1 introduces context on GTs and their fold families, emphasizing the fold architecture, canonical motifs, and catalytic mechanisms. Chapter 2 presents a review of existing literature on CS enzymes, exploring gaps in the field and extrapolating new details about CS biosynthesis and evolution. In chapter 3, we present experimental findings on the individual domains of the CS enzymes to propose a model of CS biosynthesis. In chapter 4, we examine emerging opportunities in Cryo-EM for structural studies in the CS complexes. Finally, we conclude our findings on CS enzymes in chapter 5 and highlight future research opportunities in CS biochemistry, evolution, and disease. This thesis dissertation combines our understanding of glycosyltransferases and glycosaminoglycan biosynthesis with new experimental findings to broaden the foundation of chondroitin sulfate biosynthesis in biochemistry and disease.