The use of plant growth-promoting bacteria as agricultural inoculants of plants should be encouraged because of their prominent role in biological nitrogen fixation, an increase of nutrient uptake by roots, abiotic stress mitigation, and disease control. The complex underlined mechanisms of plant and beneficial bacteria association have been increasingly studied and proteomic tools can expand our perception about fundamental molecular processes modulated by the interaction. In this study, we investigated the changes in protein expression in maize roots in response to treatment with the endophytic diazotroph Herbaspirillum seropedicae. To identify maize proteins whose expression levels were altered in the presence of bacteria, a label-free quantitative proteomic approach was used. Using this approach, we identified 123 differentially expressed proteins, of which 34 were upregulated enzymes, in maize roots cultivated with H. seropedicae. The maize root colonization of H. seropedicae modulated the differential expression of enzymes involved in the stress response and activity of enzymes related to nitrogen metabolism.