Tremendous progress has been made recently in understanding plant response to Fusarium graminearum infection. Here, the cytological aspect and molecular mechanism of maize defense to F. graminearum infection were characterized using a pair of near-isogenic lines (NIL), the resistant and the susceptible NIL. F. graminearum primarily penetrated the maize root tip and no penetration structure was found. The fungal biomass within the root correlated well with root-disease severity. Following inoculation, R-NIL and S-NIL plants significantly differed in percentage of diseased primary roots. In R-NIL roots, a fraction of exodermal cells collapsed to form cavities, and hyphae were confined to the outer exodermal cells. However, most exodermal cells shrank and turned brown, and fungi colonized the entire S-NIL root. In the R-NIL roots, the exodermal cells exhibited plasmolysis and atropous hyphal growth whereas, in the exodermal cells of the S-NIL roots, severe cellular degradation and membrane-coated, lushly grown hyphae were found. Transcriptome sequencing revealed comprehensive transcription reprogramming, reinforcement of a complex defense network, to enhance the systemic and basal resistance. This study reports a detailed microscopic analysis of F. graminearum infection on maize root, and provides insights into the molecular mechanisms underlying maize resistance to the pathogen.