Kisspeptin, a peptide product of KISS1 gene, recently identified as essential upstream gatekeeper in hypothalamic-pituitary-gonadal (HPG) axis. This study was designed to investigate the effect of immunization against kisspeptin-54 on hypothalamic-pituitary-testicular signaling pathway. A total of ten intact 56-days-old ram lambs were used and randomized into the treatment and control groups, which were, respectively immunized by kisspeptin-54 based vaccine and the empty plasmid via intramuscular route. We employed indirect enzyme-linked immunosorbent assay and quantitative real-time PCR to characterize the difference in serum kisspeptin, luteinizing hormone, testosterone hormone concentration and mRNA expression of reproductive-related genes in HPG axis across kisspeptin-54 immunized and control ram lambs. Serum kisspeptin, luteinizing hormone and testosterone concentration in the treatment group was lower (p 0.05) than that of the control group. Compared with the control group, the mRNA expression of the hypothalamic androgen receptor (AR), KISS1, G protein-coupled receptor (GPR54) and gonadotropin-releasing hormone (GnRH) was altered in the immunized group (p 0.05). Moreover, mRNA expression of pituitary luteinizing hormone beta (LHβ), follicle stimulating hormone beta (FSHβ), and GnRH receptor as well as, testicular LH receptor and FSH receptor, were remarkably lower (P 0.05) in the treatment group. We concluded that immunization against kisspeptin-54 reduced serum kisspeptin levels thereby, the normal hypothalamic-pituitary-testicular signaling pathway disrupted. This data provides a great insight for the use of kisspeptin to regulate reproduction.