ObjectiveAcupuncture has shown promise in treating neck pain. Clinical trials have shown mixed results, possibly due to heterogeneous methodologies and the lack of knowledge regarding underlying brain circuit mechanism of action. In this study, we investigated the specific contribution of the serotonergic system in treating neck pain, and the specific brain circuits involved.MethodsA total of 99 patients with chronic neck pain (CNP) were randomized to receive true acupuncture (TA) or sham acupuncture (SA) 3 times weekly for 4 weeks. Patients with CNP in each group were assessed for primary outcomes by measuring the Visual Analog Scale (VAS) and the duration of each attack; secondary outcomes were measured using the Neck Disability Index (NDI), Northwick Park Neck Pain Questionnaire (NPQ), McGill Pain Questionnaire (MPQ), Self‐rating Anxiety Scale (SAS), Self‐rating Depression Scale (SDS) and the 12‐item Short Form Quality Life Scale (SF‐12); levels of functional circuits connectivity were assessed using resting‐state functional magnetic resonance imaging in the dorsal (DR) and median (MR) raphe nucleus, before and after undergoing acupuncture.ResultsPatients receiving TA showed more extensive symptom improvement compared with SA. Regarding the primary outcomes, changes observed in the TA group were as follows: VAS = 16.9 mm (p < 0.001) and the duration of each attack = 4.30 h (p < 0.001); changes in the SA group: VAS = 5.41 mm (p = 0.138) and the duration of each attack = 2.06 h (p = 0.058). Regarding the secondary outcomes, changes in the TA group: NDI = 7.99 (p < 0.001), NPQ = 10.82 (p < 0.001), MPQ = 4.23 (p < 0.001), SAS = 5.82 (p < 0.001), SDS = 3.67 (p = 0.003), and SF‐12 = 3.04 (p < 0.001); changes in the SA group: NDI = 2.97 (p = 0.138), NPQ = 5.24 (p = 0.035) and MPQ = 2.90 (p = 0.039), SAS = 1.48 (p = 0.433), SDS = 2.39 (p = 0.244), and SF‐12 = 2.19 (p = 0.038). The modulatory effect of TA exhibited increased functional connectivity (FC) between the DR and thalamus, between the MR and parahippocampal gyrus, amygdala, and insula, with decreased FC between the DR and lingual gyrus and middle frontal gyrus, between the MR and middle frontal gyrus. Furthermore, changes in the DR‐related circuit were specifically associated with the intensity and duration of pain, and the MR‐related circuit was correlated with the quality of life with CNP.ConclusionThese results demonstrated the effectiveness of TA in treating neck pain and suggested that it regulates CNP by reconfiguring the function of the raphe nucleus‐related serotonergic system.