Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis. Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines. The reactogenicity of whole-cell pertussis vaccines is, to a large extent, derived from the lipid A moiety of the lipopolysaccharides (LPS) of the bacteria. Here, we engineered B. pertussis strains with altered lipid A structures by expressing genes for the acyltransferases LpxA, LpxD, and LpxL from other bacteria resulting in altered acyl-chain length at various positions. Whole cells and extracted LPS from the strains with shorter acyl chains showed reduced or no activation of the human Toll-like receptor 4 in HEK-Blue reporter cells, whilst a longer acyl chain increased activation. Pyrogenicity studies in rabbits confirmed the in vitro assays. These findings pave the way for the development of a new generation of whole-cell pertussis vaccines with acceptable side effects.