For identification of the underlying molecular changes in hepatic lipid metabolism in zinc deficiency, rats were force-fed a zinc-deficient diet. Subsequently DNA-microarray and proteome profiling was performed in combination with hepatic lipid analysis. Of 6200 target sequences analyzed, 268 transcripts showed altered expression levels in livers of zinc-deficient rats, with 43 genes thereof related to hepatic lipid metabolism. Northern blot analysis and quantitative real-time RT-PCR were employed to confirm changes in mRNA levels. Proteins involved in lipid metabolism were identified by proteome analysis. Functional gene clusters with uniform changes in transcript levels suggested that the pathways required for lipolysis and mitochondrial as well as peroxisomal fatty acid degradation were downregulated, whereas those needed for de novo fatty acid synthesis and triglyceride assembly were increased. Subsequent enzymatic analysis of liver tissues confirmed an almost 40% greater triacylglycerol concentration in zinc-depleted rats, as well as an altered fatty acid composition of the lipid fraction as determined by gas chromatography. Liver lipids of zinc-deficient rats had significantly greater proportions of cis-9-oleic acid, cis-11-vaccenic acid, caprylic acid, myristic acid, alpha-linolenic acid, and eicosapentaenoic acid, and significantly less stearic and arachidonic acids. These alterations in hepatic metabolism are discussed in the context of changes in mRNA and protein levels of enzymes and transporters responsible for fatty acid metabolism, sequestration, and their transcriptional control.