Fusarium verticillioides is a globally important pathogen of maize, capable of causing severe yield reductions and economic losses. In addition, F. verticillioides produces toxic secondary metabolites during kernel colonization that pose significant threats to human and animal health. Fusarium verticillioides and other plant-pathogenic fungi possess a large number of genes with no known or predicted function, some of which could encode novel virulence factors or antifungal targets. In this study, we identified and characterized the novel gene FUG1 (Fungal Unknown Gene 1) in F. verticillioides through functional genetics. Deletion of FUG1 impaired maize kernel colonization and fumonisin biosynthesis. In addition, deletion of FUG1 increased sensitivity to the antimicrobial compound 2-benzoxazolinone and to hydrogen peroxide, which indicates that FUG1 may play a role in mitigating stresses associated with host defence. Transcriptional profiling via RNA-sequencing (RNA-seq) identified numerous fungal genes that were differentially expressed in the kernel environment following the deletion of FUG1, including genes involved in secondary metabolism and mycelial development. Sequence analysis of the Fug1 protein provided evidence for nuclear localization, DNA binding and a domain of unknown function associated with previously characterized transcriptional regulators. This information, combined with the observed transcriptional reprogramming in the deletion mutant, suggests that FUG1 represents a novel class of fungal transcription factors or genes otherwise involved in signal transduction.