Early onset of puberty is a key trait underpinning economically efficient cattle production systems. Although prepubertal plane of nutrition has been positively associated with earlier onset of puberty, the underlying biochemical mechanisms remain unknown. The hypothalamus, and particularly the arcuate nucleus (ARC), has a pivotal role during this developmental stage because it is a central regulator of homeostasis, mediating various neuroendocrine and physiological functions, including feed intake and reproduction. Thus, we hypothesised that a high plane of nutrition during early calfhood would affect the transcriptional profile of hypothalamic tissue, and in particular, elicit differential expression of biochemical pathways consistent with earlier sexual development in beef heifers. Angus×Holstein-Friesian heifer calves (19±5 days of age, 51.2±7.8kg, mean±s.d.) were offered a high (HP, n=14) or moderate plane of nutrition (MP, n=15) from 3 to 21 weeks of age to achieve target growth rates of 1.2 and 0.5 kg/d, respectively. At 21 weeks of age, calves were killed and the brain recovered. The ARC region was separated from the remainder of the hypothalamus, with both tissues flash frozen and subsequently used for RNA sequencing. Differential expression of genes (DEGs) was determining using R Bioconductor package EdgeR (version 3.20.9). The resultant list of DEGs was then submitted to Ingenuity Pathway Analysis and Weighted Gene Co-expression Network Analysis (WGCNA). At slaughter, bodyweight was higher in HP calves (189.6 vs. 113.0 kg; P<0.001). Compared with calves on MP, a high plane of nutrition altered the expression of 39 and 80 transcripts in the hypothalamus and ARC, respectively (P<0.05). COL15A1, CDH17, and IL20RA were among the most upregulated transcripts in both hypothalamic tissue sections. The HP treatment induced downregulation of AGRP and NPY and upregulation of POMC in the ARC, which have previously been associated with early onset of puberty in heifers. Functional analysis highlighted the importance of DEGs in the hypothalamus and ARC related to diets, with stress-signalling pathways among the most highly dysregulated pathways in both tissues. Through pathway analysis of the ARC DEGs, a total of 7 networks were derived, including one involved in organ morphology, reproductive system development and function, and developmental disorder. Interestingly, CGA, the most upregulated DEG in the ARC and a potential upstream regulator in this brain region, was one of the most connected genes within this network. Furthermore, WGCNA revealed that expression of some hub genes in networks of co-expressed genes, previously associated with puberty in cattle, was affected by diet in the ARC (POMC, CBLN2, and CHGA) and the remainder of the hypothalamus (PENK). The results of this study indicate that an enhanced plane of nutrition during early calfhood alters the biochemical regulation of the hypothalamus consistent with advanced sexual development in the prepubertal heifer. This research was funded by Irish Department of Agriculture, Food and the Marine (RSF 13/S/515).