Introduction The hypothalamic neuropeptides kisspeptin-10 (KiSS-10), neurokinin B (NKB), and dynorphin A (Dyn A) play roles in the endocrine regulation of the hypothalamic–pituitary–ovarian (HPO) axis in puberty. Livestock’s timely attainment of sexual maturity increases reproductive efficiency and raises profitability. The pituitary relationship between these neuropeptides and gonadotropins in puberty in ewes being undercharacterised. The aim of the study was to analyse their direct effect on gonadotropin secretion by pituitary cells isolated from pubescent ewes. Material and Methods Cells were incubated in McCoy’s 5A medium, either without neuropeptides (as the control) or with 10−11, 10−10, 10−9, 10−8 and 10−7 M of KiSS-10, NKB and Dyn A. After 4, 12 and 24 h, the luteinising hormone (LH) and follicle-stimulating hormone (FSH) concentrations were analysed by ELISA using species-specific antibodies. Results Greater LH and FSH secretion was observed after the 4–24 h exposure to respective 10−11–10−8 M and 10−11–10−7 M concentrations of KiSS-10. Moreover, NKB and Dyn A applied in the concentration range elevated the secretion of both LH and FSH throughout the experiment. Dynorphin A had the most significant effect on gonadotropin secretion at all the concentrations used. In contrast, the most pronounced dose-dependent neuropeptide effect throughout the experiment on the FSH secretion was attributed to NKB. Conclusion Kisspeptin-10, NKB and Dyn A had a direct impact on gonadotropin secretion by ovine pituitary cells. However, a detailed explanation of their role in gonadotropin secretion by the anterior pituitary gland in sheep and of their impact on the regulation of the HPO axis during sexual maturation or in the pathomechanism of delayed puberty require further studies.