Metabolic stress can inhibit reproduction at least partially through altered serum gonadotropin concentrations. Kisspeptin (KP) and its receptor, Kiss1R, are critical for the function of the hypothalamic-pituitary-gonadal axis. Disruption of either KP or Kiss1R results in delayed puberty or infertility. β-Hydroxybutyrate (BHB) is a ketone body in the plasma of cattle in the negative energy state of early lactation. To determine the effects of metabolic stress on reproduction at the hypothalamus, GT1-7 cells [from mouse hypothalamic tumors which express Kiss1R and release gonadotropin releasing hormone (GnRH) in response to KP] were incubated with or without 6 mM BHB (Sigma Aldrich) for 6 h before administration of 10 nM KP (KP-10; human Metastin 45–54, Peptides International Inc.). Cells were collected at 0, 5, 15, 30, 60, 90 and 120 min following KP, and total RNA was extracted. Relative expression of GnRH (normalized to expression of GAPDH) was determined by QPCR at each time, and data were tested for effects of treatment, time, and their interaction with JMP software (version 10.0.0; SAS Inst. Inc., Cary, NC). There was an overall effect of treatment (P < 0.0001), as KP-stimulated expression of GnRH mRNA was increased by BHB [1.47 ± 0.03 (SD)] compared with those of control (1.06 ± 0.03). There was also an effect of time (P < 0.0001) and treatment x time interaction (P < 0.0001) such that KP-stimulated expression of GnRH mRNA was increased (P < 0.05) by BHB at all but one time (15 min) compared with those of control. Enhanced KP-stimulated GnRH mRNA by BHB supports published in vivo derived data indicating that feed-restricted rodents and early lactation dairy cows have greater KP-stimulated plasma gonadotropin concentrations. Utilization of GT1-7 cells, or other similar cell lines, will aid in the determination of the mechanism(s) by which BHB affects KP/Kiss1R system, providing greater understanding of the effects of metabolic stress on hypothalamic control of reproduction.