Fusarium graminearum is the causal agent of Gibberella stalk rot in maize stem, resulting maize lodging, yield, quality, and mechanical harvesting capacity. To date, little is known about the maize stem defense mechanism in response to invasion of F. graminearum. This study represents a global proteomic approach to document the infection by F. graminearum. A total of 1,894 differentially accumulated proteins (DEPs) were identified after inoculation of maize plants with F. graminearum. Functional categorization analysis indicated that proteins involved in plant-pathogen interaction were inducible at early stages of infection. We also found that the expression of proteins involved in phenylpropanoid, flavonoid, and terpenoid biosynthesis were up-regulated in response to F. graminearum infection, which may reflect a particular contribution of secondary metabolism in protection against the fungal attack in maize stem. Together, our results indicated that the defense response of maize stem to F. graminearum infection was multifaceted and involved the induction of proteins from various innate immunity related pathways, which had directive significance for molecular genetic breeding of maize disease-resistant varieties.