ENOD40 is one of the most intriguing early nodulin genes that is known to be induced very early in response to interaction of legume plants with symbiotic Rhizobium bacteria, but its function in the nodulation process is still not known. Lotus japonicus has two ENOD40 genes: LjENOD40-1 is abundantly induced in very early stages of bacterial infection or Nod factor application, whereas LjENOD40-2 is abundantly expressed only in mature nodules. We generated transgenic lines of L. japonicus with an RNAi (RNA interference) construct that expresses hairpin double-stranded RNA for LjENOD40-1 to induce sequence-specific RNA silencing. In the transgenic plants, expression of both LjENOD40-1 and -2 was significantly reduced, and no accumulation of ENOD40 transcripts was detected upon Mesorhizobium loti inoculation. The transgenic plants exhibited very poor nodulation (only 0-2 nodules per plant) and could not grow well without additional nitrogen supply. Analysis of segregation in the T(2) progeny indicated that the suppression of nodulation is perfectly linked with the presence of the transgene. Microscopic observation of the infection process using lacZ-labeled M. loti, together with expression analysis of infection-related nodulin genes, demonstrated that ENOD40 knock-down did not inhibit the initiation of the bacterial infection process. In contrast, nodule primordium initiation and subsequent nodule development were significantly suppressed in the transgenic plants. These results clearly indicate that ENOD40 is required for nodule initiation and subsequent organogenesis, but is not involved in early infection events.