The changes in the root extract protein profile of the Prunus hybrid GF 677 rootstock (P. dulcis × P. persica) grown in hydroponics as affected by Fe deficiency and short-term (24 h) Fe resupply have been studied by 2-dimensional gel electrophoresis-based techniques. A total of 335 spots were consistently found in the gels. Iron deficiency caused above 2-fold increases or >50% decreases in the relative abundance in 10 and 6 spots, respectively, whereas one spot was only detected in Fe-deficient plants. Iron resupply to Fe-deficient plants caused increases and decreases in relative abundance in 15 and 16 spots, respectively, and one more spot was only detected in Fe-resupplied Fe-deficient plants. Ninety-five percent of the proteins changing in relative abundance were identified using nanoliquid chromatography-tandem mass spectrometry. Defense responses against oxidative and general stress accounted for 50% of the changes in Fe-deficient roots. Also, a slight induction of the glycolysis-fermentation pathways was observed in GF 677 roots with Fe deficiency. The root protein profile of 24 h Fe-resupplied plants was similar to that of Fe-deficient plants, indicating that the deactivation of Fe-deficiency metabolic responses is slow. Taken together, our results suggest that the high tolerance of GF 677 rootstock to Fe deficiency may be related to its ability to elicit a sound defense response against both general and oxidative stress.