Chondroitin is a natural polymer found in the extracellular matrix and tissues of animals and is an important glycosaminoglycan derivative characterized by the repeating disaccharide units D-glucuronic acid and N-acetylgalactosamine. The sulfation patterns of these units may vary in certain regions and form chondroitin sulfate (CS), an important chondroitin derivative. The most important chondroitin sulfate derivatives such as chondroitin sulfate-A (CS-A), chondroitin sulfate-C (CS-C), chondroitin sulfate-D (CS-D), and chondroitin sulfate-E (CS-E) forms exhibit unique biological activities due to their distinct sulfation patterns that enable selective interactions in the body. Chondroitin is a polysaccharide found in many terrestrial/marine animals in the form of CS, with important biological properties including anti-osteoarthritis, anti-inflammatory, antimicrobial, anti-atherosclerotic, antioxidant, and anticancer/antitumor activities and many metabolic benefits to human health. Chondroitin and CS are of great interest due to their many in vitro/in vivo functions, and therefore, this present chapter provides information on the definitions, chemical properties, derivatives, sources, potentials/limitations, and biological functions/applications of chondroitin and focuses on microbial production of chondroitin as an alternative technique to its traditional extraction from animal tissues. Furthermore, it reviews the microorganisms currently used to produce chondroitin/CS microbially, the main biosynthetic pathways involved in their production, and the regulatory mechanisms used to regulate chondroitin/CS production. Finally, it presents the current challenges and future prospects involved in the microbial production of chondroitin/CS.