Background: Kiss-1 neurons, one of the metabolic sensors in the hypothalamic, is necessary for puberty initiating and acts through G protein-coupled receptor, known as GPR54. This study investigated the mechanism of the hypothalamic Kiss-1-GPR54 signaling pathway in high-fat diet and exercise on the growing male rats. Methods: 135 three-week-old weaned male rats were underwent high-fat diet and exercise (60–70% VO2max, 1 h/day, 5 days/ week). These mice were randomly divided into control group,

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... control and exercise group, high-fat diet group, high-fat diet and exercise group. Hypothalamic, testis, and serum samples of each group were collected at PND (postanal day)21st day (21D, early childhood), PND 43rd day (43D, puberty) and PND 56th day (56D, maturity). Immunofluorescence, Quantitative real-time PCR, hematoxylin and eosin staining, chemiluminescent immunoassays were used in studies. Analysis of variance (ANOVA) was used to analyze the effects of age (PND 21,43,56), exercise (exercise, sedentariness), and diet (high-fat, normal) on the biological indexes in ratsResults: The mRNA and protein expression of Kiss-1, GPR54 in the hypothalamic were gradually increased along with growing and peaked at PND 43, and the serum testosterone increased and peaked at PND 56. High-fat diet increased the expression of Kiss-GPR54 system in hypothalamic, while the serum testosterone decreased during different stages of growth. Exercise decreased the expression of Kiss-1 at PND 56 and increased the expression of Kiss-1at PND 43, meanwhile decreased testosterone and the deposition of lipid droplets in the testis at all age of development. Conclusions: The expression of Kiss-1-GPR54 in male rats showed fluctuated change during growth and development. High-fat diet can upregulate the expression of Kiss-1-GPR54 system in hypothalamic. Exercise can correct the adverse effect of high-fat diet on Kiss-1-GPR54 signaling pathway in hypothalamic and the function of hypothalamic-pituitary-testicular (HPT) axis, upregulate Kiss-1 at puberty and downregulate of Kiss-1 at maturity of high-fat diet male rats.