Nonhost resistance, a resistance of plant species against all nonadapted pathogens, is considered the most durable and efficient immune system in plants. To increase our understanding of the response of barley (Hordeum vulgare L.) plants to infection by powdery mildew, Blumeria graminis f. sp. Tritici, we used quantitative proteomic analysis (LC-MS/MS). We compared the response of two genotypes of barley cultivar Golden Promise, wild type (WT) and plants with overexpression of phytoglobin (previously hemoglobin) class 1 (HO), which has previously been shown to significantly weaken non-host resistance. A total of 8804 proteins were identified and quantified, out of which the abundance of 1044 proteins changed significantly in at least one of the four comparisons (‘i’ stands for ‘inoculated’)- HO/WT and HOi/WTi (giving genotype differences), and WTi/WT and HOi/HO (giving treatment differences). Among these differentially abundant proteins (DAP) were proteins related to structural organization, disease/defense, metabolism, transporters, signal transduction and protein synthesis. More proteins changed in abundance in WT than in HO after inoculation and most DAP increased in abundance.