Calcium-dependent protein kinase (CPK) family is involved in diverse functions including abiotic tolerance, however, biological functions of some CPK members have not been clarified. In our previous study, abundance of a wheat CPK protein (TaCPK34) was remarkably induced during both grain and leaf organs of wheat plants suffering from various abiotic and biotic stresses, inferring its function involved in abiotic and biotic tolerance. In present study, its function involved in abiotic tolerance was further verified. Using Agroinfiltration-mediated transient transformation, TaCPK34 protein localizes to the plasma membrane in N. benthamina leaves. Its transcripts were significantly increased during 20% PEG-induced water deficiency, and its barley stripe mosaic virus-induced silencing wheat plants exhibited more sensitive phenotype to natural drought stress, suggesting that it could play key role in response to water deficiency in wheat. Isobaric tagging for relative and absolute quantification (iTRAQ) proteomic method further revealed that, in leaves of BSMV-VIGS-induced TaCPK34 silencing wheat plants, 48 protein species exhibiting significantly altered abundance were identified during water stress condition. The identified protein species were related to diverse functions (e.g. stress and defense, carbohydrate metabolism, nucleotide metabolic, photosynthesis, transportation, protein metabolism, signal transduction, lipid and phosphate metabolism), suggesting its potential regulatory mechanism. Our results provided insights on molecular mechanism of TaCPK34 on abiotic tolerance in higher plants.