Fusarium graminearum is the main causal agent of Fusarium head blight (FHB), a destructive disease in cereal crops worldwide. Resistance to fludioxonil has been reported in F. graminearum in the field, but its underlying mechanisms remain elusive. In this study, 152 fludioxonil-resistant (FR) mutants of F. graminearum were obtained by selection in vitro. The FR strains exhibited dramatically impaired fitness, but only 7 of the 13 analyzed strains possessed mutations in genes previously reported to underlie fludioxonil resistance. Comparison between the FR-132 strain and its parental strain PH-1 using whole genome sequencing revealed no mutations between them, but transcriptome analysis, after the strains were treated with 0.5 μg/mL fludioxonil, revealed 2778 differently expressed genes (DEGs) mapped to 96 KEGG pathways. Investigation of DEGs in the MAPK pathway showed that overexpression of the tyrosine protein phosphatase FgPtp3, but not FgPtp2, enhanced fludioxonil resistance. Further analysis found that FgPtp3 interacted directly with FgHog1 to regulate the phosphorylation of Hog1, and overexpressed FgPtp3 in PH-1 could significantly suppress the phosphorylation of FgHog1 and hinder signal transmission of the HOG-MAPK pathway. Overall, FgPtp3 plays a significant role in regulating fludioxonil resistance in F. graminearum.