Enzymatic O-methylation, catalyzed by S-adenosyl-L-methionine (SAM)-dependent O-methyltranferases (OMTs), is a ubiquitous reaction, occurring in almost all living organisms. Plant OMTs are involved in the methylation of secondary metabolites, including phenylpropanoid and flavonoid compounds. Here, we used RT-PCR to isolate and characterize POMT-2 from Populus deltoides. This OMT comprises a 1095-b open reading frame that encodes a 39.7-kDa protein. BLAST results showed $87\%$ identities to an OMT from Prunus dulcis and a caffeic acid OMT from Rosa chinensis. POMT-2 was expressed in Escherichia coli as a glutathione S-transferase fusion protein, and was purified by affinity chromatography. POMT-2 transferred a methyl group of SAM to caffeic acid and 6,7-dihydroxyflavone, but showed low activities toward quercetin and kaempferol. According to its in vitro substrate preference and composition of phenolic compounds in poplar, the in vivo function of POMT-2 is probably the methylation of caffeic acid and an involvement in lignin biosynthesis.