Pueraria lobata roots accumulate 30-, 40- and 7-O-methylated isoflavones and many of these methylated compounds exhibit various pharmacological activities. Either the 40- or 7-O-methylation activity has been investigated at molecular levels in several legume species. However, the gene encoding the isoflavone 30-O-methyltransferase (OMT) has not yet been isolated from any plant species. In this study, we reported the first cDNA encoding the isoflavone 30-OMT from P. lobata (designated PlOMT4). Heterologous expressions in yeast and Escherichia coli cells showed that the gene product exhibits an enzyme activity to methylate the 30-hydroxy group of the isoflavone substrate. The transcript abundance of PlOMT4 matches well with its enzymatic product in different organs of P. lobata and in the plant roots in response to methyl jasmonate elicitation. Integration of the biochemical with metabolic and transcript data supported the proposed function of PlOMT4. The identification of PlOMT4 would not only help to understand the isoflavonoid metabolism in P. lobata but also potentially provide an enzyme catalyst for methylating existing drug candidates to improve their hydrophobicity.