Mesorhizobium loti is a member of the rhizobia and forms nitrogen-fixing symbioses with several Lotus species. Recently, it was reported that M. loti bacterial cells and their lipopolysaccharide (LPS) preparations transiently induced nitric oxide (NO) production in the roots of L. japonicus. We subsequently found that polysaccharides and the lipid A moiety were responsible for this NO induction. In this study, we elucidated the chemical structure of M. loti lipid A and characterized its NO-inducing activity in response to structural modifications. M. loti LPS were partially hydrolyzed with hydrazine or aqueous hydrofluoric acid to obtain O-deacylated or dephosphorylated LPS, respectively. The untreated and treated LPS fractions were subjected to weak acid hydrolysis to obtain lipid A fractions. The chemical structure of M. loti lipid A was elucidated by chemical composition analysis, MALDI-TOF-MS, and NMR spectra to be P-4-β-GlcNN(1-6)α-GlcNN(1-1)α-GalA, in which positions 2 and 3 of β-GlcNN are substituted for 3-acyloxy-fatty amides, and positions 2 and 3 of α-GlcNN are substituted for 3OH-fatty amides. The partial hydrolysis of lipid A appeared to reduce its NO-inducing activity. These results suggest that L. japonicus root cells recognize the lipid A structure as a means of controlling NO production.