Objective：This study aims to explore the role of ‘Brain-gut’ regulation in low-frequency auricular electro-acupuncture on maternal separation combined with enema induced visceral hypersensitivity rats. Methods：Visceral hypersensitivity was established by maternal separation combined with acetic acid enema in SD rats，which were randomly divided into the normal control（NC） group，model control（MC） group，auricular acupuncture group 1（AA1）and auricular acupuncture group 2（AA2）. Rats in the AA1 group and AA2 group were received electroacupuncture stimulation（2 Hz，30 min/d）for consecutive 10 days. The visceral sensitivity of rats was evaluated by electromyographic（EMG）to graded colorectal distension. The pathological change in distal colon were observed by HE staining. The mRNA expressions of hypothalamic corticotropin releasing factor（CRF），corticotropin releasing factor receptor1（CRF1R），corticotropin releasing factor receptor 2（CRF2R），nesfatin-1，and colonic serotonin receptor 4（5-HT4R）were detected by quantitative real-time PCR. Results：Rats in MC group show higher EMG activity，and mRNA expression of hypothalamic CRF，nesfatin-1，CRF1R，and CRF2R compared with NC group（all P < 0.05）. The colonic 5-HT4R mRNA expression in MC group shows a decreased tendency（P=0.067）. In contrast，AA treatment significantly reduced the area under the curve（AUC）（P < 0.05）. Further，compared to MC group，the mRNA expressions of hypothalamic CRF，nesfatin-1，CRF1R，and CRF2R of AA group significantly decreased（P＜0.05）while the colonic 5-HT4R mRNA expression elevated（P＜0.05）. Conclusion：Low-frequency auricular electro-acupuncture could ameliorate the visceral sensitivity in rats with visceral hypersensitivity，which might relate to the regulation of mRNA expression of CRF，nesfatin-1，CRF1R，CRF2R in hypothalamus and of 5-HT4R in colon.