Phosphorus (P) deficiency is considered as a major constraint on crop production. Although a set of adaptative strategies are extensively suggested in soybean (Glycine max) to phosphate(Pi) deprivation, molecular mechanisms underlying reversible protein phosphorylation in soybean responses to P deficiency remains largely unclear. In this study, isobaric tags for relative and absolute quantitation, combined with liquid chromatography and tandem mass spectrometry analysis was performed to identify differential phosphoproteins in soybean roots under Pi sufficient and deficient conditions. A total of 427 phosphoproteins were found to exhibit differential accumulations, with 213 up-regulated and 214 down-regulated.Taken together, identification of differential phosphoproteins not only reveled complex regulatory pathways in soybean adaptation to Pi starvation through reversible protein phosphorylation.