Gibberella ear rot (GER) caused by Fusarium graminearum (teleomorph Gibberella zeae ) is a common maize disease that not only severely reduces grain yield but also contaminates maize grain with mycotoxins. We investigated the molecular mechanism underlying the host defense responses against pathogen infection using comparative transcriptomic analysis. We injected F. graminearum spore suspensions into plants of resistant (IBM-81) and a susceptible (IBM-85) maize inbred line after pollination and performed RNA-seq 48, 72, and 96 h after inoculation. Respectively 487 and 410 differentially expressed genes (DEGs) were induced in the resistant and susceptible lines across three time points, indicating that a stronger defense response was activated in the resistant than in the susceptible line. Among them, 198 genes commonly induced in the two lines were subjected to pathway analysis, revealing that most of the DEGs were closely associated with defense and a wide range of metabolic activities. DEGs associated with pathogenesis-related protein 1 (PR1) and regulation of salicylic acid were significantly enriched during F. graminearum infection, suggesting that these DEGs play dominant roles in maize resistance to GER. Our results provide a resource for future gene discovery and facilitate elucidation of the complex defense mechanisms involved in resistance to GER.