Objective <p>The objective of this research was to clarify the effect of High Mobility Group Box 1 (HMGB1) in chronic migraine (CM).</p> Methods <p>We established a CM model by repeated intraperitoneal injection of nitroglycerin (NTG). Subsequently, Western blot was used to detect changes in HMGB1 protein expression at the trigeminal nucleus caudalis (TNC), and immunofluorescence was performed to determine HMGB1 intracellular localization. After model establishment, HMGB1 inhibitors Glycyrrhizin were administered intraperitoneally for 9 consecutive days. The paw withdrawal threshold (PWT) were measured using the electronic von Frey filament stimulation method. Additionally, changes in phosphorylated-ERK (p-ERK) and inducible nitric oxide synthase (iNOS) expression, as well as calcitonin gene-related peptide (CGRP) release levels at the TNC, were detected by WB and immunofluorescence staining.</p> Results <p>The study revealed a significant increase of HMGB1 starting on day one, which peaked on day nine. Elevated levels remained significantly high for two weeks after NTG administration(<i>p</i> &lt; 0.05). Additionally, HMGB1 was expressed in the nuclei of neurons, microglia and astrocytes. Behavioral assessments indicated a significant reduction in PWT from day five onward, indicative of mechanical hypersensitivity, which was alleviated following treatment with Glycyrrhizin(<i>p</i> &lt; 0.05).The increased expression of p-ERK and iNOS, as well as the elevated levels of CGRP, were all significantly reduced following Glycyrrhizin treatment(<i>p</i> &lt; 0.05).</p> Conclusions <p>These results show that HMGB1 plays a pivotal function in migraine by regulating neuroinflammation and central sensitization. Targeting HMGB1 may be able to mitigating chronic migraine.</p>