Escherichia coli is the workhorse for gene cloning and production of soluble recombinant proteins in both biotechnological and biomedical industries. The bacterium is also a good producer of several classes of protein-based self-assembling materials such as inclusion bodies (IBs). Apart from being a relatively pure source of protein for in vitro refolding, IBs are under exploration as functional, protein-releasing materials in regenerative medicine and protein replacement therapies. Endotoxin removal is a critical step for downstream applications of therapeutic proteins. The same holds true for IBs as they are often highly contaminated with cell-wall components of the host cells. Here, we have investigated the production of IBs in a recently developed endotoxin-free E. coli strain. The characterization of IBs revealed this mutant as a very useful cell factory for the production of functional endotoxin-free IBs that are suitable for the use at biological interfaces without inducing endotoxic responses in human immune cells.