In screening for potent antimicrobial proteins (AMPs) from plant seeds, we had purified a heat-stable AMP, LJAMP2, from the seeds of a medicine herb, motherwort (Leonurus japonicus Houtt). In an in vitro assay, the protein can inhibit the growth of both fungi and bacteria. Then a cDNA encoding LJAMP2 was cloned by the rapid amplification of cDNA ends based on the N-terminal amino acid sequence determined. The deduced amino acid sequences of this cDNA show similarity to plant non-specific lipid transfer proteins. Northern blotting assay revealed that this nsLTP-like gene, designated LJAMP2, was expressed in seeds. Overexpression of LJAMP2 in tobacco enhanced resistance to the fungal pathogen Alternaria alternata and the bacterial pathogen Ralstonia solanacearum, significantly, while no visible alteration in plant growth and development. Our data confirm the antifungal and antibacterial function of LJAMP2 from motherwort seeds and suggest the potential of LJAMP2 in improving disease resistance in plants.