This study was performed to determine the optimal condition for Lentinula edodes JMI-10079 mycelium cultivation on the root of Polygonum multiflorum. We also analyzed the proximate composition, total amino acids, and minerals in the root of P. multiflorum cultivated with L. edodes JMI-10079 mycelia. The optimal temperature and pH for L. edodes JMI-10079 mycelium cultivation on the P. multiflorum root were 25oC and pH 5–6 respectively, whereas the optimal carbon and nitrogen sources were glucose and maltose, respectively. The content of crude protein, crude fat, and ash in the P. multiflorum root cultivated with L. edodes JMI-10079 mycelia was higher than that in the uncultivated P. multiflorum root. The content of crude fiber was the highest in the control. Total amino acid analysis revealed that the contents of total amino acids and total essential amino acids were increased by higher root of P. multiflorum concentration.