Fusarium graminearum is the causal agent of gibberella ear rot in maize ears, resulting in yield losses due to mouldy and mycotoxin-contaminated grain. This study represents a global proteomic approach to document the early infection by F. graminearum of two maize inbreds, B73 and CO441, which differ in disease susceptibility. Mock- and F. graminearum-treated developing kernels were sampled 48 h post-inoculation over three field seasons. Infected B73 kernels consistently contained higher concentrations of the mycotoxin deoxynivalenol than the kernels of the more tolerant inbred CO441. A total of 2067 maize proteins were identified in the iTRAQ analysis of extracted kernel proteins at a 99% confidence level. A subset of 878 proteins was identified in at least two biological replicates and exhibited statistically significantly altered expression between treatments and/or the two inbred lines of which 96 proteins exhibited changes in abundance >1.5-fold in at least one of the treatments. Many proteins associated with the defense response were more abundant after infection, including PR-10 (PR, pathogenesis-related), chitinases, xylanase inhibitors, proteinase inhibitors, and a class III peroxidase. Kernels of the tolerant inbred CO441 contained higher levels of these defense-related proteins than B73 kernels even after mock treatment, suggesting that these proteins may provide a basal defense against Fusarium infection in CO441.