Fibromyalgia (FM) is a complex, chronic, widespread pain syndrome that can cause significant health and economic burden. Emerging evidence has shown that neuroinflammation is an underlying pathological mechanism in fibromyalgia. Toll-like receptors (TLRs) are key mediators of the immune system. TLR4 is expressed primarily in microglia and regulates downstream signaling pathways, such as MyD88/NF-κB and TRIF/IRF3. It remains unknown whether electroacupuncture (EA) has therapeutic benefit in attenuating fibromyalgia pain and what role the TLR4 pathway may play in this effect. We compared EA with sham EA to eliminate the placebo effect due to acupuncture. We demonstrated that intermittent cold stress significantly induced an increase in mechanical and thermal FM pain in mice (mechanical: 2.48 ± 0.53 g; thermal: 5.64 ± 0.32 s). EA but not sham EA has analgesic effect on FM mice. TLR4 and inflammatory mediators-related molecules were increased in FM mice’s thalamus, medial prefrontal cortex, somatosensory cortex, and amygdala, indicating neuroinflammation and microglial activation. These molecules were reduced by EA but not sham EA. Furthermore, a new chemogenetics method was used to precisely inhibit SSC activity displayed an anti-nociceptive effect through TLR4 pathway. Our results imply that the analgesic effect of EA is associated with TLR4 downregulation. We provide novel evidence that EA can modulate the TLR4 signaling pathway, revealing potential therapeutic targets for FM pain.