Mechanisms that regulate the milk synthetic response to nutrient supply in lactating dairy cows remain largely unexplained. The objective of this study was to evaluate short- and long-term changes in expression of mammary genes related to secretory cell turnover and milk-synthesizing activity per cell in lactating Holstein dairy cattle subjected to restricted feeding. Pairs of cows (234 ± 27 DIM, n = 7) were blocked by date and average daily milk yield and fed either 100 or 60% of ad libitum intake for 14 d. The feed restriction treatment commenced with 16 h of no access to feed. Milk production dropped 4.8 kg/d by d 1 of restriction and remained at that level until d 14 (P < 0.01). On d 1, plasma glucose and BHBA concentrations did not differ between treatment groups (P > 0.67), but FA concentrations were 2 times higher (P < 0.01) in restricted cows. There were no differences in these metabolite concentrations between treatment groups on d 13 (P > 0.18). Mammary mRNA expression of milk protein genes and genes related to protein synthesis and secretion were not affected after 16 h of feed withdrawal (P > 0.10), but expression and protein abundance of cyclin D1 were downregulated 56 and 42% (P ≤ 0.04), respectively. After 14 d, cyclin D1 expression in mammary tissue was no longer low (P = 0.32) but expression of the pro-apoptotic DNA damage-inducible transcript 3 (aka CHOP) was elevated 69% (P = 0.04). There were no differences between treatments in mammary parenchymal DNA mass or proportions of proliferating and apoptotic cells on d 14 (P > 0.37). However, parenchymal tissue and protein mass were 24 and 29% lower, respectively, in restricted versus unrestricted cows (P = 0.03) and the glands produced 45% less milk daily per gram of parenchymal DNA. Results suggest that both mammary cell number and activity per cell are acutely regulated within 16 h of a change in total dietary nutrient supply, and that chronic changes in milk yields can be sustained without chronic changes in cell proliferation or apoptosis rates.