Peroxisomes are organelles that are involved in a number of important cellular metabolic processes, including the β-oxidation of fatty acids, biosynthesis of secondary metabolites, and detoxification of reactive oxygen species (ROS). In this study, the role of peroxisomes was examined in Fusarium graminearum by targeted deletion of three genes (PEX5, PEX6, and PEX7) encoding peroxin (PEX) proteins required for peroxisomal protein import. PEX5 and PEX7 deletion mutants were unable to localize the fluorescently tagged peroxisomal targeting signal type 1 (PTS1)- and PTS2-containing proteins to peroxisomes, respectively, whereas the PEX6 mutant failed to localize both fluorescent proteins. Deletion of PEX5 and PEX6 resulted in retarded growth on long-chain fatty acids and butyrate, while the PEX7 deletion mutants utilized fatty acids other than butyrate. Virulence on wheat heads was greatly reduced in the PEX5 and PEX6 deletion mutants, and they were defective in spreading from inoculated florets to the adjacent spikelets through rachis. Deletion of PEX5 and PEX6 dropped survivability of aged cells in planta and in vitro due to the accumulation of ROS followed by necrotic cell death. These results demonstrate that PTS1-dependent peroxisomal protein import mediated by PEX5 and PEX6 are critical to virulence and survival of F. graminearum.