Performance of dairy cows can be influenced by early life nutrient supply. Adipose tissue is diet sensitive and an important component in that process as it is involved in the regulation of energetic, reproductive and immunological functions. However, it is not clear how early life nutrition alters the molecular regulation of adipose tissue in calves and potentially adult individuals. This study aimed at determining how differences in pre-weaning nutrient supply alter gene expression profiles and physiology in omental adipose tissue. A total of 12 female Holstein calves were fed two levels of milk replacer supply: a restricted amount of 11.72 MJ of metabolizable energy (ME) intake per day (n = 6) or an enhanced amount of 1.26 MJ ME intake per kg of metabolic body weight (BW0.75), resulting in supply from 17.58 to 35.17 MJ ME intake per day (n = 6). All calves had ad libitum access to a commercial calf starter and water. Analysis of the transcriptome profiles at 54 ± 2 days of age revealed that a total of 396 out of 19,968 genes were differentially expressed (DE) between groups (p < 0.001, FDR < 0.1). The directional expression of DE genes through Ingenuity Pathway Analysis showed that an enhanced nutrient supply alters adipose tissue physiology of pre-weaned calves. Several biological functions were increased (Z-score > +2), including Lipid Metabolism (Fatty Acid Metabolism), Cell Cycle (Entry into Interphase, Interphase, Mitosis and Cell Cycle Progression), Cellular Assembly and Organization (Cytoskeleton Formation and Cytoplasm Development) and Molecular Transport (Transport of Carboxylic Acid). These changes were potentially orchestrated by the activation/inhibition of 17 upstream regulators genes. Our findings indicate that adipose tissue of calves under an enhanced nutrient supply is physiologically distinct from restricted calves due to an increased development/expansion rate and also a higher metabolic activity through increased fatty acid metabolism.