Abstract Purpose: To find the expression of KISS-1 and G protein-coupled receptor 54 in rats testis from PND 21st to 56th. Method: 128 three-week-old weaned rats underwent high-fat diet and exercise (60%–70% VO2max, 1 h/day, 5 days/week) intervention and were randomly divided into group C, CE, HC, or HE. Sample time points were set on the PND 21st, 35th, 43rd, and 56th. The testicular testosterone and the mRNA content, and protein content of KISS-1 and GPR54 in testis tissue were detected by ELISA, RT-qPCR, and Western blotting. Result: (1) The protein of KISS-1 and GPR54 increased gradually during the growing period. KISS-1 mRNA peaked at 35D and GPR54 peaked at 43D. (2) High-fat diet affected the expression of the KISS-1/GPR54 system in rat testis and reduced the expression level of KISS-1 protein. (3) 60%–70% VO2max exercise decreased the KISS-1/GPR54 expression level. Exercise intervention improved testicular development in rats with a high-fat diet.Conclusion: The expression of KISS-1/GPR54 increased during the growing period. High-fat diet can downregulate the protein and gene expression of KISS-1/GPR54 and change the expression trend. 60%–70% VO2max exercise decreased the expression of KISS-1/GPR54, which may be involved in the effects of exercise on high-fat dietary sex hormone disorders.