An organ unique to mammals, the mammary gland develops 90% of its mass after birth and experiences the pregnancy-lactation-involution cycle (PL cycle) during reproduction. To understand mammogenesis at the transcriptomic level and using a ribo-minus RNA-seq protocol, we acquired greater than 50 million reads each for the mouse mammary gland during pregnancy (day 12 of pregnancy), lactation (day 14 of lactation), and involution (day 7 of involution). The pregnancy-, lactation-and involution-related sequencing reads were assembled into 17344, 10160, and 13739 protein-coding transcripts and 1803, 828, and 1288 non-coding RNAs (ncRNAs), respectively. Differentially expressed genes (DEGs) were defined in the three samples, which comprised 4843 DEGs (749 up-regulated and 4094 down-regulated) from pregnancy to lactation and 4926 DEGs (4706 up-regulated and 220 down-regulated) from lactation to involution. Besides the obvious and substantive upand down-regulation of the DEGs, we observe that lysosomal enzymes were highly expressed and that their expression coincided with milk secretion. Further analysis of transcription factors such as Trps1, Gtf2i, Tcf7l2, Nupr1, Vdr, Rb1, and Aebp1, Let7,, and mir-15 has enabled us to identify key regulators in mammary gland development and the PL cycle. mouse mammary gland, mammogenesis, transcriptome, rmRNA-seq, miRNAs, transcription factors Citation: