<jats:title>Abstract</jats:title><jats:p>We have defined the complete structure and assessed the biological activity of the lipopolysaccharide (LPS) from <jats:italic>Alkalimonas delamerensis</jats:italic>, an alkaliphilic bacterium isolated from soda lakes in China and East Africa. The structural determination, which was achieved by the use of chemical, spectroscopic and spectrometric approaches, indicates a novel Rough type lipopolysaccharide that is rich in negatively charged groups in the lipid A inner core region. The proinflammatory activity of the pure lipopolysaccharide has also been defined by testing the LPS as a stimulant in human HEK293 cells, which express the CD14/MD2/TLR4 complex (HEK293‐hTLR4), and in bone marrow‐derived macrophages.