cDNA for a major arbuscular mycorrhiza (AM)-inducible phosphate (Pi) transporter of Lotus japonicus, LjPT3, was isolated from Glomus mosseae-colonized roots. The LjPT3 transcript was expressed in arbuscule-containing cells of the inner cortex. The transport activity of the gene product was confirmed by the complementation of a yeast mutant that lacks high-affinity Pi transporters. In contrast to most AM-inducible Pi transporters thus far reported, LjPT3 has an amino acid sequence that has much in common with those of other members of the Pht1 family of plant Pi transporters, such as StPT3 of potato. To understand better the physiological role of this AM-inducible Pi transporter, knockdown transformants of the gene were prepared through hairy root transformation and RNA interference. Under Pi-limiting conditions, the transformants showed a reduction of Pi uptake via AM and growth retardation. The transformants also exhibited a decrease in G. mosseae arbuscules. Additionally, when Mesorhizobium loti was inoculated into the knockdown transformants in combination with G. mosseae, necrotic root nodules were observed. Based on these findings, we consider that the genetically engineered host plants had monitored insufficient Pi uptake via AM or low expression of LjPT3, excluding the existing fungi and rhizobia and/or preventing further development of the fungal and nodule structures.