In order to characterize the significance of sulfur (S) nutrition in protein expression under iron (Fe)-deficient conditions, gel-based proteomic analysis was performed with the leaves of Brassica napus exposed to S and Fe combined treatments: sufficient in S and Fe (+S/+Fe, control), sufficient S but Fe deprived (+S/-Fe), deprived S but sufficient Fe (-S/+Fe), and deprived S and Fe (-S/-Fe). The resulting data showed that 15 proteins were down-regulated due to production of oxidative damage as indicated by H2O2 and O2 -1 localizations and due to leaf chlorosis in leaves in S-deprived leaves either in presence (-S/+Fe) or absence of Fe (-S/-Fe), whereas these down-regulated proteins were well expressed in the presence of S (+S/-Fe) compared to control (+S/+Fe). In addition, two proteins were up-regulated under S-deprived condition in presence (-S/+Fe) and absence of (-S/-Fe) Fe. The functional classification of these identified proteins was estimated that 40 % of the proteins belong to chloroplast precursor, and rest of the proteins belongs to hypothetical proteins, RNA binding, secondary metabolism and unknown proteins. On the other hand, five protein spots from S deprived (-S/+Fe) and ten spots from Fe deprived (-S/-Fe) conditions were absent, whereas they were well expressed in presence of S (+S/-Fe) compared to control plants (+S/+Fe). These results suggest that sulfur nutrition plays an important role in alleviating protein damage in Fe-deficient plants and adaptation to Fe-deficiency in oilseed rape. © 2013 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.