Protein and fat content in milk replacer influences tissue growth and metabolism; however, the intrinsic influence on performance has yet to be fully evaluated. We hypothesized that feeding milk replacer varying in dietary energy content would elicit differential expression of genes within pathways associated with growth and metabolism. The objectives of this study were to (1) identify transcripts differentially expressed in tissues related to growth and metabolism in pre-weaned dairy heifers, and (2) determine the growth and metabolic pathways influenced by these transcripts. Pre-weaned Holstein heifers (n = 12; age 6 d ± 0.02) were randomly assigned to 1 of 2 milk replacer diets: Enhanced (E; 28% Fat, 25% CP; n = 6), or Restricted (R; 20% Fat, 20% CP; n = 6). After 8 wks, samples from longissimus dorsi (LD), adipose (A), and liver (L) tissues were collected, snap frozen and stored at −80°C. Libraries were constructed from extracted RNA for RNA-Seq analyses. Average daily gain (ADG) and gain-to-feed ratio (G:F) were calculated for each calf. Analysis of ADG and G:F was performed using a PROC GLM in SAS with diet as the main effect; E calves had increased ADG and G:F. RNA-Seq analysis was performed using CLC Genomics Workbench and the Robinson and Smith Exact Test was used to identify differentially expressed genes between diets. There were 238 differentially expressed genes in A, 227 in LD, and 40 in L. Of the differentially expressed genes, 10 appeared in at least 2 tissues. PANTHER was used to identify functional categories of differentially expressed genes. The majority of genes were associated with metabolic processes (A = 112, 26.7%; L = 16, 32.0%; LD = 81, 34.0%) or cellular processes (A = 93, 22.1%; L = 13, 26.0%; LD = 73, 30.7%). In E calves, upregulated genes included those regulating NADH dehydrogenation (LD = 17, A = 5; i.e., ND1, ND4), gluconeogenesis (LD = 2, A = 6; i.e., ALDOB, PCK2), and cell proliferation (LD = 2, A = 3; i.e., GADD45A, CDKN1A). This change in regulation of cell cycle and ATP synthesis in response to dietary energy could explain the change in ADG between diets.