Background <p>Chronic insomnia disorder (CID) is characterized by dysregulation in brain function and is closely associated with neuroinflammation. Although acupuncture has been shown to improve insomnia symptoms, its underlying mechanisms, particularly at both the macro brain connectivity and corresponding molecular levels, remain unclear</p> Methods <p>Forty-eight CID patients were randomly assigned to either an acupuncture group or a waitlist group. Clinical data and resting-state functional magnetic resonance imaging (rs-fMRI) data were collected before and after the intervention. Changes in brain connectivity were analyzed using fMRI to assess global brain connectivity (GBC) in each group. Gene expression data from the Allen Human Brain Atlas were utilized to identify important genes contributing to these acupuncture-induced GBC changes. Gene set enrichment analysis was performed to annotate the molecular biological processes involved.</p> Results <p>In the acupuncture group, fMRI analysis revealed decreased regional GBC in key regions, such as the pallidum and prefrontal cortex, correlating with symptom relief. In contrast, the waitlist group showed increased regional GBC without symptom relief. Gene set enrichment analysis revealed that specific genes associated with astrocytes and neuroinflammation-related biological processes were linked to the acupuncture-induced changes in GBC. The neuroinflammation-informed GBC-transcriptomic signatures induced by acupuncture were further validated by their significant correlation with reductions in IL-6 levels as insomnia symptoms improved.</p> Conclusion <p>Acupuncture may remodel brain functional connectivity by regulating neuroinflammation-related pathways, thereby improving insomnia symptoms.</p> Graphical abstract <p></p>