Technical Abstract: Iron deficiency is a yield-limiting factor with major implications for field crop production in one-third of the world's agricultural areas, especially those with high soil CaCO3. A two-dimensional gel electrophoresis proteomic approach was combined with a study on the riboflavin synthesis pathway, including qPCR and riboflavin determination, to investigate Fe-deficiency responses in Medicago truncatula plants grown with and without CaCO3. Iron deficiency caused a de novo accumulation of DMRLs and GTPcII; proteins involved in riboflavin biosynthesis, as well as marked increases in root riboflavin concentrations and in the expression of four genes from the riboflavin biosynthetic pathway. Two novel changes were the increased accumulation of proteins related to N recycling and protein catabolism. Other identified changes were consistent with previously shown increases in glycolysis, TCA-cycle, and stress-related processes. All effects were more marked in the presence of CaCO3. The riboflavin synthesis pathway was up-regulated (at the genomic, proteomic and metabolomic levels) under both Fe deficiency treatments, especially in the presence of CaCO3. Results also indicate that N recycling occurs in M. truncatula upon Fe deficiency, possibly constituting an additional anaplerotic N and C source for the synthesis of secondary metabolites, carboxylates, and others.