ABSTRACT: In the field, abiotic stresses are rarely applied individually. Crops are often subjected to a combination of stresses. To date, no study has been performed on the proteomic investigation of the response of common wheat to a combination of drought and cold stresses. In this study, wheat seedlings exposed to drought-cold stress for 24 h showed inhibited growth, increased lipid peroxidation, relative electrolyte leakage, and soluble sugar contents. To determine the wheat protein response to drought-cold stress, iTRAQ-based quantitative proteomic and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were employed to determine the proteomic profiles of the roots and leaves of wheat seedlings exposed to drought-cold stress conditions. We identified 250 and 258 proteins with significantly altered abundance in the roots and leaves, respectively. These proteins were classified into several main groups, as follows: protein metabolism, stress/defense, carbohydrate metabolism, lipid metabolism, transcription-related processes, energy production, cell-wall and cytoskeleton metabolism, membrane and transportation, signal transduction, other metabolic processes, and unknown biological processes. Nine proteins were simultaneously presented in both roots and leaves exposed to drought-cold stress, and the majority of proteins identified differed from one another and displayed differently altered abundance. These findings uncovered organ-specific differences in adaptation to drought-cold stress. Exogenous abscisic acid (ABA) application conferred the plant with protection against drought-cold stress and significantly increased catalase and peroxidase enzyme activities, as well as the transcription of glutathione S-transferase and other 11 sample genes in the roots or leaves, respectively. These results suggested that ABA is a potentially vital factor that contributes to the drought-cold signaling pathway and a promising target for growth recovery. Furthermore, VIGS (virus-induced gene silencing)-treated plants generated for three candidate protein genes TaGRP2, CDCP and WCOR410c were subjected to drought-cold limitation, they showed more serious droop and wilt, increased rate of relative electrolyte leakage, and reduced relative water content (RWC) compared to viral control plants. These results may indicate that TaGRP2, CDCP and WCOR410c play important roles in conferring drought-cold tolerance in wheat. These findings can provide useful insights into the molecular mechanisms of drought-cold responses in higher plants.