Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. However, the pathogenic mechanism and host’s responses after DTMUV infection remain poorly understood. During this study, total mRNA sequencing (RNA-Seq) analysis was used to detect the global gene expression in DEFs at various time points after DTMUV infection. We identified 326 genes altered significantly at all time points, and these genes were dynamically enriched in multifarious biological processes, including apoptosis, innate immune response, DNA replication, cell cycle arrest and DNA repair. Next, the results showed that apoptosis was induced and the proportion of apoptosis increased with time, and pro-apoptotic molecules caspases were activated. The RNA-seq data analysis further revealed that most pro-apoptosis and anti-apoptosis genes were early continually responsive, and the genes involved in both intrinsic and extrinsic apoptotic pathways were initiated. Further, the considerably enriched immune-relevant pathways were involved in apoptosis process, and protein-protein interactions (PPIs) analysis showed that IL6, STAT1, TNFAIP3, CFLAR and PTGS2 may be key regulators of DEFs apoptosis. In conclusion, this study not only contributes to understanding the underlying mechanism of DEFs infection with DTMUV, but also provides new insights into targets screening for antiviral therapy.