Aspergillus flavus is an opportunistic fungal pathogen that produces carcinogenic aflatoxin, a serious constraint for food safety and human health. In this study, to better understand the molecular mechanism/s of peanut resistance to A. flavus growth and aflatoxin accumulation, comparative proteomic analysis was performed in two contrasting peanut genotypes, variety JL 24 (susceptible) and its near-isogenic resistant transgenic derivative expressing an alfalfa defensin gene. Several resistance proteins associated with secondary metabolic pathways were strongly induced in the resistant genotypes including phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase, chalcone synthase, resveratrol synthase, flavanone-3-hydroxylase, lipoxygenase, diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase, beta-ketoacyl-ACP-reductase, monoacylglycerol acyltransferase, and diacylglycerol acyltransferase, indicating their roles in resistance. Besides, several putative susceptibility-associated proteins were revealed providing knowledge on potential candidate target genes for precise breeding interventions for aflatoxin mitigation. This is the first study to demonstrate comparative proteomics analysis in Aspergillus-peanut interaction using contrasting near-isogenic lines to elucidate the underlying molecular mechanisms of resistance.