Carbohydrate modification is a common phenomenon in nature. Many carbohydrate modifications such as some epimerization, O-acetylation, O-sulfation, O-methylation, N-deacetylation, and N-sulfation, take place after the formation of oligosaccharide or polysaccharide backbones. These modifications can be categorized as carbohydrate post-glycosylational modifications (PGMs). Carbohydrate PGMs further extend the complexity of the structures and the synthesis of carbohydrates and glycoconjugates. They also increase the capacity of the biological regulation that is achieved by finely tuning the structures of carbohydrates. Developing efficient methods to obtain structurally defined naturally occurring oligosaccharides, polysaccharides, and glycoconjugates with carbohydrate PGMs is essential for understanding the biological significance of carbohydrate PGMs. Combined with high-throughput screening methods, synthetic carbohydrates with PGMs are invaluable probes in structure-activity relationship studies. We illustrate here several classes of carbohydrates with PGMs and their applications. Recent progress in chemical, enzymatic, and chemoenzymatic syntheses of these carbohydrates and their derivatives are also presented.