<p>Painful peripheral neuropathy afflicts 30% of diabetic patients and necessitates improved therapies. Acacetin, a natural O-methylated flavonoid, has been observed to exhibit antidepressant properties in preclinical investigations. As antidepressants are established therapies for enduring neuropathic pain, this study aimed to investigate the potential antinociceptive efficacy of acacetin in managing neuropathic pain associated with diet-induced type 2 diabetes, and to probe mechanism(s). C57BL6J male mice were fed with high-fat diet for eight weeks to induce type 2 diabetes. The resulting development of neuropathic pain was quantified by assessing sensitivity to mechanical stimuli (the von Frey test) and cold stimuli (the acetone test), indicative of neuropathic allodynia. Chronic acacetin therapy (5, 15 and 45 mg/kg) not only ameliorated the established symptoms of mechanical and cold allodynia in type 2 diabetic mice, but also decelerated pain development in preventive regimen at lower doses (2, 6 and 18 mg/kg). Although acacetin did not impacted metabolic features (levels of body weight, food intake, blood glucose, glucose tolerance, glycosylated haemoglobin and insulin) in diabetic mice, it corrected defects of mitochondrial bioenergetics in dorsal root ganglion neurons, mitigated oxidative stress in pain-associated tissues, and improved sciatic nerve conduction velocity and blood flow. Notably, the pain-alleviating actions of acacetin were modulated by pharmacological agents targeting mitochondrial bioenergetics and redox balance. These findings uncover the antinociceptive effect of acacetin, which is mechanistically associated with its capacity to enhance mitochondrial function and mitigate oxidative stress, in mice with diet-induced type 2 diabetes.</p>