Involution in the dairy cow is critical for maximal milk yield in the next lactation. We characterized the mammary gland transcriptome during early involution through the description of the major pathways and biological function of expressed genes. Holstein cows (n = 6), were housed in free stall shaded barns with fans and soakers during the entire dry period (~46 d). Mammary gland biopsies were collected at −3, 3, 7, and 14 d relative to dry-off. Total RNA extraction, amplification, library preparation, and sequencing were performed following Illumina HiSeq 3000 protocol. Sequencing reads were mapped to the bovine reference genome (bosTau7) using Tophat. The resulting alignments were used to reconstruct transcript models using Cufflinks. Ingenuity Pathway Analysis was used to determine top canonical pathways and biological functions at each time point. The p-value was calculated using Fisher’s t-test from the ratio of genes in the data set to the genes in the Ingenuity Knowledge base for a specific function or pathway. Overall, we identified 63, 112, 78 and 90 canonical pathways for d −3, 3, 7 and 14, respectively (−log P-value > 10). The top 10 pathways across time points were similar but the level of significance differs (i.e., protein ubiquitination, EIF2, mTOR, eIF4, p70S6K, integrin, Rho family GTPases, glucocorticoids receptor, PI3K-AKT, B-cell receptor and PTEN signaling; −log P-value > 15). The top 10 biological functions across time points were similar but the level of significance also differs (i.e., cell death, cell proliferation, necrosis, apoptosis, expression and transcription of RNA, response to viral infection, organization of cytoplasm, metabolism of proteins, cell movement; −log P-value > 54). Notably, cell proliferation and cell death functions involve 260 and 269 more molecules on d3 than d-3, the organization of cytoplasm 47 more molecules on d7 than d3, and the function cell movement 17 more molecules at d14 relative to d7. Top biological functions reiterate the importance of cell death and proliferation during mammary involution, and the top canonical pathways reveal novel cellular signaling molecules that may play important roles in early mammary gland involution.