Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused huge economic losses to the duck industry in China since 2010. Moreover, the infection has spread rapidly, resulted in a potential public health concern. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we used RNA-Seq to detect the gene changes in DF-1 cells infected and mock-infected with DTMUV. A total of 663 differentially-expressed genes (DEGs) were identified in DTMUV-infected compared with mock-infected DF-1 cells at 24 h post-infection (hpi), among which 590 were up regulated and 73 were down regulated. Gene Ontology analysis indicated that the DEGs were mainly involved in cellular process, immune system processes, metabolic processes, and signal-organism process. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were mainly involved in several signaling pathways such as Toll-like receptor signaling, Jak-STAT signaling, RIG-I-like receptor signaling and AGE-RAGE signaling pathway. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the sequencing data. To our knowledge, this study is the first to analyze the transcriptomic change in DF-1 cells following DTMUV infection. We believe that our research provides useful information in better understanding the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity.