Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.