Medicago truncatula constitutes a good model for Strategy I plants, since when this plant is challenged with Fe shortage the most important root physiological responses induced by Fe deficiency are developed, including the yellowing of root tips. A better understanding of the mechanisms involved in root adaptation to Fe deficiency in M. truncatula may strengthen our ability to enhance Fe efficiency responses in other plant species, especially in different agronomically relevant legumes. Riboflavin concentration, phosphoenolpyruvate carboxylase (EC 4.1.1.31) and Fe reductase activities, and acidification capacity have been determined in M. truncatula roots at different time points after imposing Fe deficiency. Root riboflavin concentrations increased with Fe deficiency and concomitantly MtDMRL was upregulated at the transcriptional level, supporting a role for flavins in the Fe deficiency response. Root Fe reductase and phosphoenolpyruvate carboxylase activities as well as acidification capacity were higher in roots of Fe-deficient than in control plants, and the corresponding genes, MtFRO1, MtPEPC1 and MtHA1 were also upregulated by Fe deficiency. Expression of these genes and their corresponding physiological activities followed different patterns over time, suggesting the existence of both transcriptional and post-transcriptional regulation.