Nitrogen nutrition is one of the major factors limiting the growth and production of crop plants. Limited information on proteome changes occurring in response to nitrogen amount have been available up to now. We used 2-DE to investigate proteome differences between two triticale varieties and the changes caused by nitrogen nutrition deficit in the flag leaf tissue. Some physiological features, such as the number of tillers per plant, SPAD index, dry weight, and protein content were measured previous to the proteomic analysis. Statistical analysis identified 29 differential protein spots in the selected pairwise comparisons of experimental conditions and correlated with the expression cluster revealed by the principal component analysis. The 29 protein spots were subjected to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) to deduce their possible functions. Many of these changes referred to enzymes involved in photosynthesis, metabolic pathways implicated in the balance of the energy, and redox status of the cell. This work provides a first characterization of the proteome changes that occur in response to nitrogen deficit in flag leaves of triticale plants.