Histological and functional changes associated with involution in the mammary gland are partly regulated by changes in gene expression. At 42 d postpartum, Holstein cows underwent a period of 5 d during which they were milked 1X daily until complete cessation of milking. Percutaneous mammary biopsies (n = 5/time point) were obtained on d 1, 5, 14, and 21 relative to the start of 1X milking for transcript profiling via qPCR of 57 genes associated with metabolism, apoptosis/proliferation, immune response/inflammation, oxidative stress, and tissue remodeling. Not surprisingly, there was clear downregulation of genes associated with milk fat synthesis (FASN, ACACA, CD36, FABP3, SCD) and lipid-related transcription regulation (SREBF1, SREBF2). Similar to milk fat synthesis-related genes, those encoding proteins required for glucose uptake (SLC2A1), casein synthesis (CSN2, CSN3), and lactose synthesis (LALBA) decreased during involution. Unlike metabolic genes, those associated with immune response and inflammation (C3, LTF, SAA3), oxidative stress (GPX1, SOD2), and pro-inflammatory cytokine signaling (SPP1, TNF) increased to peak levels by d 14 from the start of 1X milking. These adaptations appeared to be related with tissue remodeling as indicated by upregulation of proteins encoding matrix proteinases (MMP2), IGFBP3, and transcriptional regulation of apoptosis/cell proliferation (MYC). In contrast, the concerted upregulation of STAT3, TGFB1, and TGFB1R during the first 14 d was suggestive of an activation of these signaling pathways probably as an acute response to regulate differentiation and/or mammary cell survival upon the onset of a marked pro-inflammatory and oxidative stress response induced by the gradual reduction in milk removal. Results suggest a central role of STAT3, MYC, PPARG, SREBF1, and SREBF2 in regulating concerted alterations in metabolic and cell survival mechanisms, which were induced partly via oxidative stressed-triggered inflammation and the decline in metabolic activity.