Fusarium graminearum is the most devastating pathogen of Fusarium head blight of cereals, stalk and ear of maize, and it has recently become a potential threat for soybean as maize-soybean strip relay intercropping is widely practiced in China. To elucidate the pathogenesis mechanism of F. graminearum on intercropped soybean which causes root rot, transcriptional profiling of F. graminearum at 12, 24, and 48 h post-inoculation (hpi) on soybean hypocotyl tissues was conducted. In total, 2313 differentially expressed genes (DEGs) of F. graminearum were annotated by both KEGG pathway and Gene Ontology (GO) analysis. Among them, 128 DEGs were commonly expressed at three inoculation time points while the maximum DEGs were induced at 24 hpi. In addition, DEGs were also rich in carbon metabolism, ribosome and peroxisome pathways which might contribute to carbon source utilization, sexual reproduction, virulence and survival of F. graminearum when infected on soybean. Hence, this study will provide some basis for the deep understanding the pathogenesis mechanism of F. graminearum on different hosts and its effective control in maize-soybean strip relay intercropping systems.