Physiological and endocrine studies on sexual development in animals and effects of acupuncture on sexual development are limited. Therefore, we investigated the effect of electro-acupuncture (EA) on the arcuate nucleus (Arc) and release of gonadotropin-releasing hormone (GnRH) in animals at different developmental stages. In Experiment 1, EA stimulation (30 Hz) was performed for 30 min per day in EA group of rabbits for 48 days, while the control group (mature rabbits) was not given EA. Arc discharges in those two groups were measured after the 48-day treatment. Arc discharge was also measured in the pre-pubertal group (as control) without EA treatment. Then, all three groups were treated with transient EA for 30 min and Arc discharges were determined again. In Experiment 2, EA (3 Hz) at the same acupoints or non-acupoints as that in the rabbits was performed for 20 min per day in different developmental group of Sprague-Dawley rats for 10 days. GnRH mRNA expression in the hypothalamus of rats was determined using RT-PCR and real-time PCR. The serum sexual hormone, sperm count, and body weight was measured. The results showed that the Arc discharge (P<0.01), testosterone (T) (P<0.01) and sperm count (P<0.01) in male rabbits were reduced by repeated EA. However, the body weight of rabbits was not changed after EA compared to the control in Experiment 1. In Experiment 2, GnRH mRNA expression in rats of the early pubertal group (EPG) and adult group (AG) were significantly depressed after repeated EA at acupoints (P<0.01). The sexual hormones were negatively influenced by repeated EA during puberty. Sperm count was reduced significantly after repeated EA at time of puberty (P<0.01). Repeated EA did not influence body weight of rats (P>0.01) and structures of the gonadial tissues during development. The results suggested that repeated EA is a good option that can be considered for regulating the function of the hypothalamus-pituitary-gonad (HPG) axis during puberty.