Copper (Cu) is an essential micronutrient for humans and animals. Moderate Cu deficiency during pregnancy can be serious for the baby, leading to both short and long term consequences such as connective tissue abnormalities, skeletal defects and abnormalities in cardiac ultra structure. During pregnancy, the placenta regulates the transfer of micronutrients to the growing fetus. Little is known about the molecular adaptation in the placenta as a response to Cu deficiency. We compared the protein expression profile from placenta of rats fed diet low in Cu (0.75 mg Cu/kg diet) to that of placenta of rats fed control diet (5 mg Cu/kg diet). 12 Female Rowett Hooded Lister rats were weaned onto control diet. They were then randomly assigned to one of the two dietary treatments: control (n=6) or Cu deficient (n=6) diet for 4 weeks prior to mating and throughout pregnancy up to gestation day 21 where the placentas were isolated. Protein extracts were separated by 2-D gel electrophoresis, analyzed by PDQuest and subsequently by mass spectrometry and sequencing. Principal component analysis of the spot density values revealed that 24% of all variance in the data set was accounted for by the first principal component and an additional 15% was accounted for by the second principal component. There was a clear separation of the spots into two groups corresponding to the two treatments. Spot intensities were analyzed by Student’s t test at 5% significance to determine differences in responses between the control and Cu-deficient group. Cu deficiency during pregnancy resulted in differential expression of 60 proteins in the rat placenta. These were identified as proteins involved in, for example, signal transduction, protein synthesis, calcium signaling, iron metabolism and structural proteins, enzymes and chaperones. Using pairwise correlation analysis (Pearson correlation coefficient > 0.6) we identified two clusters of proteins in the data set. One cluster of proteins included acute phase alpha-1 protein, transferrin and serotransferring, liver regeneration related protein, aconitase hydratase, 3-alpha hydroxysteroid dehydrogenase, tropomyosin 1, myosin regulated light chain 2-A, 26 protease regulatory subunit S10B, ribosomal protein RS 40K, voltage-dependent anion-selective protein 1, beta actin, keratin type II, proliferating nuclear antigen and guanine nucleotide-binding protein beta-2 chain. Of these, 7 proteins were over expressed and the rest down regulated in the placenta of Cu deficient animals as compared to controls. The other cluster identified comprised mainly of secreted proteins which were all down regulated. In this study, we have, by using proteomics techniques, provided novel insights into the mechanism by which Cu deficiency during pregnancy may affect the developing fetus.