Derivatives of 4-hydroxy-6-methyl-2-pyrone（2-pyrone） are actively involved in plant resistance to insects and pathogens, and are considered as potential renewable chemical platforms for phloroglucinol and 1,3,5-triamino-2,4,6-trinitrobenzene. 2-Pyrone synthase (2PS), one of the type Ⅲ polyketide synthases (PKSs), is the key enzyme to produce 2-pyrone. In this study, a novel Pc2PS was isolated from Polygonum cuspidatum Sieb. et Zucc., a traditional Chinese herb used for the treatment of cough, suppurative conditions, and hypertension. The Pc2PS shared 54%–56% amino acid sequence identity with 2PSs of other species. Enzymatic activity analysis showed that Pc2PS could catalyze the condensation of one molecule of acetyl-CoA with two molecules of malonyl-CoA at almost the same efficiency as that of three molecules of malonyl-CoA alone for production of 4-hydroxy-6-methyl-2-pyrone. Thus, the presence or absence of acetyl-CoA in the reaction mixture did not affect catalytic efficiency. This catalytic property of Pc2PS differs from that of previously reported Gh2PSs, which catalyzed malonyl-CoA condensation to form the same products as Pc2PS but at a lower rate in the absence of acetyl-CoA. In addition, we determined the kinetics of Pc2PS with malonyl-CoA alone as the substrate for the first time. Pc2PS showed tissue-specific expression in P. cuspidatum; Pc2PS was predominantly expressed in the root, whereas few transcripts were detected in the leaf. This study enriches the known diversity of 2PS proteins, and provides a novel genetic resource for 2-pyrone biosynthesis.