Gram-negative bacteria have an outer membrane that is positioned at the frontline of the cell’s interaction with the environment and that serves as a barrier against noxious molecules including many antibiotics. This protective function mainly relies on lipopolysaccharide, a complex glycolipid located in the outer leaflet of the outer membrane. In this chapter we will first summarize lipopolysaccharide structure, functions and biosynthetic pathway and then we will discuss how it is transported and assembled to the cell surface. This is a remarkably complex process, as amphipathic lipopolysaccharide molecules must traverse three different cellular compartments to reach their final destination.