Growth hormone (GH) is an essential factor in enhancing the productivity of animals. In ruminants, L-aspartate (L-Asp) stimulates the secretion of GH; however, the effect of D-Asp on GH remains unknown. Here, we examined the effect of D-Asp on GH secretion in wethers. Blood GH, insulin, adrenaline, noradrenaline, non-esterified fatty acid (NEFA), and glucose concentrations were evaluated in response to the intravenous infusion of a high-dose (0.1 mmol/kg/min) of D-Asp for 20min. Further, concentrations of these biomolecules were evaluated when a low-dose (0.05 mmol/kg/min) of D-Asp was continuously infused intravenously for 20min. Finally, the direct effect of D-Asp on GH secretion was determined using cultured sections of the anterior pituitary tissue from wethers. Infusion of the high-dose of D-Asp markedly increased blood GH concentrations (P < 0.05), resulting in an increase in the area under the curve (AUC). Plasma GH concentrations and AUC also increased in response to infusion of a low D-Asp dose. Infusion of a high and low D-Asp dose caused a prolonged reduction in plasma insulin concentrations, and the AUC was lower (P < 0.05). Plasma NEFA concentrations gradually increased after the end of D-Asp infusion, with a low D-Asp dose infusion resulting in significantly higher concentrations at 90min (P < 0.05). Plasma adrenaline, noradrenaline, and glucose concentrations did not show significant changes despite differences in the dose of D-Asp. Although D-Asp treatments stimulated GH secretion in the cultured sections of pituitary tissues, the effect was not significant. These results suggest that D-Asp stimulates the secretion of GH in wethers through not only a direct action on the pituitary gland but also through another pathway of GH stimulation.