Nutritional and metabolic exposure during critical periods of prenatal and neonatal development in animals and humans may induce long-term effects on health status in later life. The aim of this study was to investigate the effects of neonatal treatment with β-hydroxy-β-methylbutyrate (HMB) during the first 3 weeks of life on programming of skeletal development in sheep. The study was performed on 12 male sheep divided into two groups. While the control group was treated with placebo, the lambs in the HMB group received the calcium salt of β-hydroxy-β-methylbutyrate at a daily dose of 0.1 g (kg body weight)−1. The assessment of growth hormone (GH), insulin-like growth factor-1 (IGF-1) and biochemical bone turnover markers in serum was performed in 21- and 130-day-old animals. Post mortem, volumetric bone mineral density, morphological and mechanical properties were determined in femur and lumbar vertebrae. Neonatal treatment with HMB increased serum concentrations of bone-specific alkaline phosphatase, osteocalcin, GH and IGF-1 in 21-day-old lambs by 125.2, 93.8, 71.8 and 70.9%, respectively (P≤ 0.05). The C-terminal telopeptide of type I collagen (CTX) concentration was increased in 130-day-old animals from the HMB group by 33.1% (P= 0.03). Furthermore, HMB administration improved bone weight, volumetric bone mineral density and bone morphological and mechanical properties of femur and lumbar spine. In conclusion, this study showed long-term beneficial effects of neonatal treatment with HMB on programming of peripheral and axial skeleton properties that were mediated by a transient improvement of somatotrophic axis function and acceleration of bone metabolism.