Abstract: Background　Migraine is a highly prevalent neurovascular disorder. Previous studies have demonstrated that the dopamine system in the medial orbitofrontal cortex (MO) exhibits functional abnormalities in migraine patients. As a critical component of the mesolimbic dopamine system, the dopamine D2 receptor (Drd2) within the MO plays an important role in pain perception and emotional modulation. Nevertheless, the specific role and regulatory mechanism of Drd2-positive neurons in the MO in the pathogenesis and progression of migraine remain to be clarified. Objective　This study aimed to investigate the regulatory role of Drd2⁺ neurons in the MO in migraine-related hyperalgesia and anxiety-like behaviors, as well as to preliminarily explore the underlying mechanisms. Methods　The study employed intraperitoneal injection of nitroglycerin (NTG, 10 mg/kg) or normal saline to establish acute migraine model mice and control group mice, respectively. The mechanical pain thresholds of the plantar region and peri-orbital area were measured using Von Frey filaments. Anxiety-like behavior in both groups was assessed using the open field test. In vivo fiber photometry calcium imaging was utilized to real-time record the dynamic changes in calcium activity of Drd2 ⁺ neurons in the MO before and after NTG injection. Chemogenetic approaches were applied to modulate neuronal activity in the model group mice, which were further divided into an activation group, an inhibition group, and a control group. The mechanical pain thresholds (plantar and peri-orbital) and anxiety-like behavior were subsequently examined and compared among these three groups. Results　Two hours after intraperitoneal NTG injection, the mechanical pain thresholds of the plantar region (P＜0.05) and peri-orbital area (P＜0.01) in mice were significantly decreased. Concurrently, the time spent in the central zone of the open field was reduced (P＜0.05). Fiber photometry results revealed a sustained decreasing trend in the calcium activity of Drd2⁺ neurons in the MO cortex following NTG injection. The calcium signals significantly decreased compared to the baseline level at 30-40 minutes post-injection (P＜0.01) and reached their lowest point at 90-100 minutes (P＜0.001). Following chemogenetic inhibition of Drd2⁺ neuron activity in the MO cortex, the mechanical pain thresholds of the plantar region (P＜0.01) and peri-orbital area (P＜0.001) in mice were significantly increased. Furthermore, their time spent in the central zone of the open field was significantly prolonged (P ＜0.05). In contrast, specific activation of these neurons did not produce a significant effect on the aforementioned two pathological phenotypes (P＞0.05). Conclusion　The decreased activity of Drd2⁺ neurons in the MO is involved in mediating the pathogenesis and progression of migraine-related hyperalgesia and anxiety-like behaviors; specific inhibition of the activity of these neurons can effectively alleviate the aforementioned pathological phenotypes.