Frankia and actinorhizal plants exchange signals in the rhizosphere leading to specific mutual recognition of partners and nitrogen-fixing nodule organogenesis. Frankia soli strain NRRL B-16219, from the Elaeagnus specificity group, colonizes the root tissues of its actinorhizal host through direct intercellular penetration of root epidermis cells and cortex. Here, we studied the early proteogenomic response of strain NRRL B-16219 to treatment with root exudates from compatible Elaeagnus angustifolia, and incompatible Ceanothus thyrsiflorus and Coriaria myrtifolia, host plants grown in nitrogen depleted hydroponic medium. Next-generation proteomics was used to identify the main Frankia proteins differentially expressed in response to the root exudates. No products of the nod genes present in B-16219 were detected. Proteins specifically upregulated in presence of E. angustifolia root exudates include those connected to nitrogen fixation and assimilation (glutamate synthetase, hydrogenase and squalene synthesis), respiration (oxidative phosphorylation and citric acid cycle pathways), oxidative stress (catalase, superoxide dismutase, and peroxidase), proteolysis (proteasome, protease, and peptidase) and plant cell wall degrading proteins involved in the depolymerization of celluloses (endoglucanase, glycosyltransferase, beta-mannanases, glycoside hydrolase and glycosyl hydrolase). Proteomic data obtained in this study will help link signaling molecules/factors to their biosynthetic pathways once those factors have been fully characterized.