For ruminants, dietary protein is the first limiting component to the utilization of low-quality forage. Throughout gestation, low-protein intake may result in prenatal programming that causes various metabolic disturbances and physiological modulations to dams and their developing embryos. We studied the effect of long-term low-protein diet (LPD) on physiological, biochemical, and molecular parameters of the energy status in gestating beef cows. LPD resulted in significant reductions in feed intake and heart rate and promoted a negative retained energy status already after 3 weeks. Elevated levels of plasma creatinine and non-esterified fatty acids indicate endogenous degradation of fat and protein as a response to the demands in energy and nitrogen. Increasing levels of β-hydroxybutyrate confirmed the negative energy status obtained by the physiological measurements. At the molecular level, subcutaneous fat, Hsp90, Hsp70, and proteasome subunits decreased significantly after 3 months on LPD, in parallel with an increase of adipocyte fatty acid-binding protein. These results may indicate a decrease in turn-over of proteins, at the cost of induced lipolysis, and suggest that the response to protein deprivation, when examined in an energy-storing tissue, includes downregulation of the constitutive heat shock proteins involved in the protein degradation pathway of energy production and upregulation of tissue-specific genes such as those involved in energy production from fat degradation.