Phosphorus (P) is a macronutrient with structural and regulatory functions, essential for energy transfer. Under limited P availability, plant cells respond to internal signals, adjusting their metabolic pathways in order to reorganize physiological priorities. This work was conducted with the aim to explore the initial changes following P deprivation, before neither growth nor photosynthesis was strongly affected. The first unifoliate leaves of nine-day-old soybean plants (Glycine max, cv. Williams 82) were analyzed. Plants grown hydroponically under control conditions (+P, nutrient solution with 500 ?M H2PO4?) were compared with those restricted in phosphate (-P, without H2PO4? in the nutrient solution). No visible symptoms of P-deficiency, such as changes in pigment intensity and leaf number or size, were observed up to 48 h of P deprivation. Neither fresh weight nor shoot/root FW ratio was affected in plants as a consequence of early P-restriction. Shotgun proteomic analyses of leaves from plants exposed to 24 h of P-deprivation, revealed that a total of 202 proteins were exclusively detected and 232 increased their relative abundance under –P conditions. The proteins affected belong mainly to the catalytic activity group according to Gene Ontology Consortium including proteins like 6-phosphogluconate dehydrogenase (from pentose phosphate pathway), pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit alpha, alpha-1,4 glucan phosphorylases (from carbohydrate metabolic processes), and other enzymes involved in metabolic processes such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and fructose-bisphosphate aldolase (from gycolisis), and nitrate reductase (from nitrogen assimilation). Early events in leaves also involve higher levels of the bioactive molecule nitric oxide (NO), detected employing confocal laser microscopy, accompanied with a parallel increase in nitrate reductase activity. Furthermore, the presence of nitrated proteins was detected using shotgun liquid chromatography–mass spectrometry (LCeMS/MS). This post-translational protein modification, affected proteins mainly related to photosynthesis (chlorophyll a–b binding protein, ribulose bisphosphate carboxylase), and some other metabolic processes (glutamine synthetase, methionine synthase, lipoxygenase). This methodology allowed the confirmation of nitration sites in proteins previously described as putatively nitrated. Taken together the data presented suggest important metabolic changes, including nitric oxide metabolism, during the first hours of P deprivation.