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Abstract: Dihydromyricetin (DMY) is a principle bioactive component of the wild plant Ampelopsis grossedentata and possesses multiple pharmacological effects. However, the mechanism of its biological activity has not received much attention. In this study, the inhibitory mechanism of DMY was explored. Experiments were conducted to investigate the inhibitory effects of DMY on the proline metabolism of Vibrio parahaemolyticus by measurements of proline contents and proline dehydrogenase (PDH) activity. With DMY treatment, an increase in proline content and decrease in PDH activity were observed. Furthermore, molecular docking studies were used to confirm interaction mechanism of DMY with PDH. The results shown that DMY can interact with primary amino acid residues located within the active hydrophobic pockets of PDH, leading to decrease of PDH activity. The normal metabolism of proline was interfered by DMY, resulting in molecular damage and even death of V. parahaemolyticus cells. Practical applications: The antimicrobial mechanism of dihydromyricetin (DMY) on Vibrio parahaemolyticus at the molecular level is reported for the first time in this study. The molecular docking simulation provided supportive data for DMY‐induced inhibition by allowing us to predict the binding site in the active site pocket of proline dehydrogenase. The data can be useful for future studies that involve evaluation of antibacterial mechanism of natural antimicrobial agents against foodborne pathogens and provide an insight into the structural and antibacterial properties of phenolic compounds. [ABSTRACT FROM AUTHOR]