BACKGROUND: Chronic migraine is a prevalent and potentially debilitating neurological disorder that is often comorbid with mental health conditions (such as anxiety and depression), but the underlying mechanisms linking these conditions remain poorly understood. Indoleamine 2,3-dioxygenase 1 (IDO1) has been implicated in inflammatory processes, including neuroinflammation and pain. However, its role as a link between neuroinflammation and pain sensitization in chronic migraine is not well defined. METHODS: Male mice were used to establish a model of chronic migraine by recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg). Using pharmacological approaches, transgenic strategies and adeno-associated virus (AAV) intervention, we investigated the role of IDO1 in pain sensitization and migraine-related mood disorders in an NTG-induced chronic migraine mouse model. We employed a combination of immunoblotting, immunohistochemistry, three-dimensional reconstruction, RNA sequencing, electrophysiology, in vivo fiber photometry, and behavioral assays to elucidate the underlying mechanisms involved. RESULTS: Our findings demonstrated that pharmacological inhibition and genetic knockout of IDO1 significantly alleviated pain sensitivity in a chronic migraine model. Neuronal activity in the anterior cingulate cortex (ACC) was evaluated with in vitro c-Fos immunostaining as well as in vivo fiber photometry, and a shift in the excitation/inhibition (E/I) balance toward excitation was observed through whole-cell patch clamp recording. Notably, IDO1 expression was increased in the ACC, and AAV-mediated IDO1 knockdown in the ACC rescued pain sensitivity, electrophysiological E/I balance changes, and anxiety-like behavior in chronic migraine model mice. Furthermore, IDO1 regulated microglial activation and pruning of neuronal synapses in the ACC. IDO1's microglial pruning function appears to be mediated through the interferon (IFN) signaling pathway, and the behavioral changes induced by IDO1 knockdown in the ACC could be reversed by activating this pathway. CONCLUSIONS: Our findings revealed that microglial IDO1 in the ACC drives pain sensitization and anxiety in chronic migraine, highlighting IDO1 as a potential therapeutic target for chronic migraine treatment.