This study was aimed to observe the effects of acupuncture combination with medicated thread moxibustion ofZhuang nationality medicine on neurotransmitters in rats with sleep deprivation. A total of 80 SPF grade healthy Wistar rats were randomly divided into the control group, model group, treatment group and diazepam group with 20 rats in each group. The establishment of insomnia model was through sleep deprivation in the model group, treatment group and diazepam group. Based on the treatment of the model group, the treatment group was treated by acupuncture combination with medicated thread moxibustion ofZhuang nationality medicine, once a day for continuous 14 days. Intragastric administration of diazepam according to 3 mg·kg-1 body weight was given in the diazepam group. While intragastric administration of the same volume normal saline was given to the control group once a day for continuous 14 days. And then, the levels of orexin, NE, 5-HT, CRH and ACTH were detected. The expressions of orexin mRNA and c-Fos were observed. The results showed that compared with the control group, the levels of orexin, ACTH, 5-HT, NE and CRH increased or obviously increased in the model group with significant difference (P < 0.05, orP< 0.01). The expressions of orexin mRNA and c-Fos in the model group were increased or obviously increased with significant difference (P < 0.05, orP< 0.01). Compared with the model group, the levels of orexin, ACTH, 5-HT, NE and CRH reduced or obviously reduced with significant difference in the treatment group (P < 0.05, orP < 0.01). The expressions of orexin mRNA and c-Fos were reduced or obviously reduced with significant difference in the treatment group (P <0.05, orP < 0.01). It was concluded that acupuncture combination with medicated thread moxibustion ofZhuang nationality medicine took an effect on the treatment of insomnia by mediating neurotransmitters associated with hypothalamic-pituitary-adrenal axis (HPA axis) through orexin, which was probably related with its therapeutic mechanism.