Technical Abstract: Infection of the developing maize (Zea mays L.) ear with the pathogenic fungus Aspergillus flavus, which produces the carcinogen aflatoxin, is a serious agricultural problem causing significant economic losses worldwide. The rachis (or cob) is an important structure that fungus uses to spread within ear and infect the kernels. Because of this and its importance in maize reproduction, we performed the first high-throughput proteomic analysis of developing rachis from the A. flavus resistant maize inbred line Mp313E. The rachis proteome was investigated using two complementary proteomic approaches, two-dimensional electrophoresis (2-DE) and two-dimensional liquid chromatography (2-D LC) coupled to tandem mass spectrometry and database search. A total of 1,037 proteins were identified, of which 266 proteins (25.7%) were identified by both approaches whereas 277 (26.7%) and 494 (47.6%) were unique to 2-DE and 2-D LC, respectively. A 2-D proteome reference map was also established. Functional classification of identified proteins based on Gene Ontology annotation revealed that young Mp313E rachis contains proteins of diverse molecular activities that are involved in various metabolic processes. Most importantly, we determined that 13.4% of its proteome is constituted of stress-responsive, defense-related and secondary metabolism proteins that may contribute to the overall resistance to A. flavus colonization.