Background: Soybean downy mildew (SDM), caused by Peronospora manshurica (Pm), is a major fungal disease in soybean. To date, little is known regarding the defense mechanism at molecular level and how soybean plants response to Pm infection. In this study, differential gene expression in SDM-resistant (HR) and SDM-susceptible (HS) genotype was analyzed by RNA-seq to identify differentially expressed genes (DEGs) following Pm infection. Results: Of a total of 55,017 genes mapped to the soybean reference genome sequences, 2581 DEGs were identified. Clustering analysis of DEGs revealed that these genes could be grouped into 8 clusters with distinct expression patterns. Functional annotation based on gene ontology (GO) and KEGG analysis indicated they involved in diverse metabolism pathways. Of particular interest were the detected DEGs participating in SA/ROS and JA signalling transduction and plant/pathogen interaction. Conclusion: Totally, 52 DEGs with P value < 0.001 and log(2) fold change > 2 or < - 2 upon fungal inoculation were identified, suggesting they were SDM defense responsive genes. These findings have paved way in further functional characterization of candidate genes and subsequently can be used in breeding of elite soybean varieties with better SDM-resistance.