Lactation plays the crucial role in mammals’ life. Uncovering the transcriptome signature of lactation process helps to understand the molecular basis of milk production. To identify the genes that express differentially between early and late lactation, publicly available microarray transcriptomic datasets of dairy cattle were investigated and the array results were validated by a next-generation sequencing dataset (RNA-Seq data from sheep). Limma and edgeR packages were used for the analysis of the microarray and RNA-Seq datasets, respectively. Five common differentially expressed genes (DEGs), namely glutathione s-transferase mu 3 (GSTM3 ), EGF containing fibulin-like extracellular matrix protein 1(EFEMP1 ), fibulin 1(FBLN1 ), gelsolin (GSN ), and fibrinogen-like 2 (FGL2 ), were identified. The involvement of EFEMP1 in the lactation process has been reported for the first time. The identified DEGs are involved in the development of the immune system and cell differentiation of the mammary gland. A gene ontology network analysis revealed the key role of the GSN gene in the regulation of two important functions of actin nucleation and barbed-end actin filament capping. The gene ontology enrichment analysis showed that the function of calcium ion binding is statistically (P < 0.05) enriched by the identified transcriptomic signature. The approach presented in this study provides an integrative framework for finding the signature of the lactation process by utilizing global transcriptome data, gene ontology (GO) regulatory network, and enrichment analysis.