The expression of genes and their regulation during lactation in Ghezel sheep breed remains less understood. To explore the underlying molecular mechanism of the lactation process in the mammary gland, transcriptome profiles of Iranian fat-tailed Ghezel sheep breed milk at two stages, before (BF) and after peak (AF) stages of lactation were investigated. Functional impacts of differentially expressed genes (DEGs) between BF and AF stages were surveyed using Gene Ontology (GO) and Protein-Protein Interaction (PPI) network analysis. Totally, 75 DEGs were identified between BF and AF stages of lactation. The RNA-Seq results were validated by Q-RT-PCR. Gene ontology of DEGs mainly enriched in metabolic process and oxidative phosphorylation. PPI network analysis also highlighted the contribution of peroxisome proliferator-activated receptors (PPAR) signaling, oxidative phosphorylation and metabolic pathways in the lactation process. Intriguingly, the genes involved in fat metabolism dominantly down-regulated at AF stage. Our results provide new insight into transcriptional changes and add to growing body of knowledge on the lactation process in fat-tailed sheep breeds.