The liver of dairy cows is involved in signaling the current metabolic state to the brain via metabolites and nerval afferents to control and adjust feed intake. Feed deprivation may result in mobilization of body reserves favoring hepatic steatosis. While the overall metabolic changes are well characterized specific regulatory mechanisms are not readily understood. In order to identify molecular events associated with metabolic adaptation and the control of energy homeostasis, liver specimens from six ad libitum fed and six feed deprived cows were analyzed for selected metabolites, for the activation of AMP kinase, and for regulatory/regulated proteins using 2-D gel electrophoresis and MALDI-TOF-MS. Feed deprivation increased total liver fat, and the calcium content, as well as augmented AMPK phosphorylation while it decreased the contents of protein, glucose, glycogen, and cholesterol when expressed as % of dry matter. Among 34 differentially expressed proteins identified, we found downregulation of proteins associated with fatty acid oxidation, glycolysis, electron transfer, protein degradation and antigen processing, as well as cytoskeletal rearrangement. Proteins upregulated after feed deprivation included enzymes of the urea cycle, fatty acid or cholesterol transport proteins, an inhibitor of glycolysis, and previously unknown changes in calcium signaling network. Direct correlation was found between expression of glycolytic enzymes and glucose/glycogen content, whereas inverse correlation exists between expression of beta-oxidative enzymes and total liver fat content. In conclusion, the regulatory response of identified proteins may help to explain development and consequences of hepatic lipidosis but also offers novel candidates potentially involved in signaling for maintaining energy homeostasis.