Mycolic acids are major and specific long-chain fatty acids that represent essential components of the Mycobacterium tuberculosis cell envelope. They play a crucial role in the cell wall architecture and impermeability, hence the natural resistance of mycobacteria to most antibiotics, and represent key factors in mycobacterial virulence. Biosynthesis of mycolic acid precursors requires two types of fatty acid synthases (FASs), the eukaryotic-like multifunctional enzyme FAS I and the acyl carrier protein (ACP)-dependent FAS II systems, which consists of a series of discrete mono-functional proteins, each catalyzing one reaction in the pathway. Unlike FAS II synthases of other bacteria, the mycobacterial FAS II is incapable of de novo fatty acid synthesis from acetyl-coenzyme A, but instead elongates medium-chain-length fatty acids previously synthesized by FAS I, leading to meromycolic acids. In addition, mycolic acid subspecies with defined biological properties can be distinguished according to the chemical modifications decorating the meromycolate. Nearly all the genetic components involved in both elongation and functionalization of the meromycolic acid have been identified and are generally clustered in distinct transcriptional units. A large body of information has been generated on the enzymology of the mycolic acid biosynthetic pathway and on their genetic and biochemical/structural characterization as targets of several antitubercular drugs. This chapter is a comprehensive overview of mycolic acid structure, function, and biosynthesis. Special emphasis is given to recent work addressing the regulation of mycolic acid biosynthesis, adding new insights to our understanding of how pathogenic mycobacteria adapt their cell wall composition in response to environmental changes.