Using a deep serial analysis of gene expression (DeepSAGE) sequencing approach, we profiled the transcriptional response of Fusarium graminearum to tebuconazole, a most widely used azole fungicide. By comparing the expression of genes in F. graminearum treated and untreated with tebuconazole, we identified 324 and 155 genes showing more than a 5-fold increase and decrease, respectively, in expression upon tebuconazole treatment. These genes are involved in a variety of cell functions including egrosterol biosynthesis, transcription, and cellular metabolism. The validity of DeepSAGE results were confirmed by real-time PCR analysis of expression of 20 genes with different expression levels in the DeepSAGE analysis. The results from this study provide useful information in understanding the mechanisms for the responses of F. graminearum to azole fungicides.