We constructed T-DNA insertional lines of a model legume, Lotus japonicus, using a multifunctional vector for gene and exon activation tagging. The vector had the CaMV 35S promoter together with two additional enhancer elements, the start codon, and splice donor and acceptor sites facing the left border, in anticipation of the activation of T-DNA flanking genes and forced expression of flanking exons. The improved transformation technique yielded more than 3,500 lines, including 45 dominant mutant candidates with abnormal phenotypes with respect to aerial parts, nodules, and roots. Among the 44 selected lines, one copy of T-DNA was inserted into the genome of 37 lines (84%). The T-DNA flanking regions of seven lines were isolated by thermal asymmetric interlaced (TAIL)-PCR or reverse transcription (RT)-PCR, and the corresponding genomic clones were analyzed. The transcripts of four genes adjacent to T-DNA out of 11 genes tested were increased in the T(1) generation, demonstrating that gene and exon activation effects by the newly developed tagging vector are heritable. The T-DNA insertional population of L. japonicus will provide legume-specific dominant mutants.