Endotoxin refers lipopolysaccharide that constitutes the outer leaflet of the outer membrane of most Gram-negative bacteria. Lipopolysaccharide is comprised of a hydrophilic polysaccharide and a hydrophobic component known as lipid A which is responsible for the major bioactivity of endotoxin. Lipopolysaccharide can be recognized by immune cells as a pathogen-associated molecule through Toll-like receptor 4. Most enzymes and genes related to the biosynthesis and export of lipopolysaccharide have been identified in Escherichia coli, and they are shared by most Gram-negative bacteria based on available genetic information. However, the detailed structure of lipopolysaccharide differs from one bacterium to another, suggesting that additional enzymes that can modify the basic structure of lipopolysaccharide exist in bacteria, especially some pathogens. These structural modifications of lipopolysaccharide are sometimes tightly regulated. They are not required for survival but closely related to the virulence of bacteria. In this chapter we will focus on the mechanism of biosynthesis and export of lipopolysaccharide in bacteria.