Chronic metabolic diseases, particularly insulin resistance (IR) and diabetes, pose significant global health challenges. This study introduces a novel hydroxylated dihydromyricetin (DHM) derivative, 8-hydroxy-DHM (H-DHM), produced via microbial fermentation using Beauveria bassiana. Notably, hydroxylation significantly enhances the efficacy of DHM in glucose consumption, glycogen synthesis, and glucose transport, while inhibiting gluconeogenesis in an IR-HepG2 cell model. This indicates that hydroxylation of DHM can enhance its regulation of glucose metabolism. Mechanistic investigations reveal that H-DHM regulates the JNK/PI3K/AKT signaling pathway by reducing inflammation, oxidative stress, and endoplasmic reticulum stress. These findings highlight the potential of hydroxylated DHM as a promising candidate for dietary and clinical interventions in IR management. Furthermore, this research provides new insights into the modification of natural flavonoids through microbial fermentation, presenting an innovative strategy for managing and preventing chronic metabolic diseases.