To observe the effect of liver-soothing and mental-activity-regulating (LSMAR) needling on behavior reactions and contents of serum calcitonin gene-related peptide (CGRP), substance P (SP), interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in migraine rats, so as to explore its mechanism underlying relief of migraine. Thirty-two male Wistar rats were randomly divided into control, model, LSMAR needling and conventional needling groups (n=8 rats in each). The migraine model was established by hypodermic injection of glyceryl trinitrate (5 mg/kg) at the napex. Acupuncture preconditioning was conducted once daily for 8 days before modeling. Acupuncture needles were inserted into "Baihui" (GV 20), and bilateral "Fengchi" (GB 20), "Neiguan" (PC 6) and "Taichong" (LR 3), manipulated for a while and retained for 30 min. The rats' behavioral changes (times of head scratching, tail-biting, cage-crawling, and to- and fro-movement in 30 min) were scored (one symptom = one point) before modeling, after modeling, and after intervention. The concentrations of CGRP, SP, IL-1 β, TNF-α in serum were assayed by enzyme-linked immunosorbent assay (ELISA). After modeling, the behavioral scores were significantly increased in the migraine model rats relevant to the control group (P<0.05). Whereas after the treatment, the behavior scores were considerably decreased during 60-90 min and 120-150 min of the measuring period in both LSMAR and conventional needling groups compared with the model group (P<0.05). The concentrations of CGRP, SP, IL-1 β and TNF-α were significantly higher in the model group than in the control group (P<0.05), and notably lower in both LSMAR and conventional needling groups than in the model group (P<0.05). The therapeutic effect of LSMAR was markedly superior to that of conventional needling in down-regulating the concentrations of CGRP, SP, IL-1 β and TNF-α (P<0.05). Acupuncture preconditioning can effectively relieve pain in migraine rats possibly by decreasing the concentrations of CGRP, SP, IL-1 β and TNF-α in the peripheral blood, and the therapeutic effect of LSMAR is evidently superior to that of conventional needling.