The initiation and maintenance of lactation are complex phenomena governed by biochemical and endocrine processes in the mammary gland (MG). Despite extensive studies at the genetic, physiological and morphological levels, only limited data are available concerning the gene alterations of lactating and non-lactating bovine MG. To determine how the gene expression profile vary during lactation compared with the dry period, we used RNA-sequencing based transcriptomics to identify how genes isolated from lactating and non-lactating bovine MG tissue samples differed. A total of 918 differentially expressed genes (DEG, including 674 up-regulated and 244 down-regulated) were identified in lactating group versus nonlactating group. These up-regulated DEG included genes associated with various macromolecular metabolic processes, and appear to promote the increased metabolic activity related to milk synthesis and secretion. Functional analysis indicated that these DEG were primarily involved in initiation, maintenance, and involution of lactation, and included genes related to glycolysis/gluconeogenesis, the TCA cycle, oxidative phosphorylation, aminoacyl-tRNA biosynthesis, protein folding/synthesis/transport, fatty acid biosynthesis and cell growth. The results also suggested that non-lactating cows may have had depressed milk protein synthesis because these animals had decreased capacity for protein synthesis, enhanced proteolysis, inefficient energy generation and reduced cell growth. The 45 identified DEG associated with the process of protein synthesis were further validated by RT-PCR. This work provided some gene-associated insights to facilitate further investigation of the mechanisms underlying lactation in dairy cows.