Muscovy duck reovirus (MDRV) causes high morbidity and mortality in ducklings. However, the molecular basis for pathogenesis of this virus remains poorly understood, and the complete genome sequence of Muscovy duck is lacking. Here we report the transcriptome profile of Muscovy ducks in response to MDRV infection. RNA sequencing technology was employed to obtain a representative complement of transcripts from the spleen of ducklings, and then differential gene expression was analyzed between MDRV-YB strain infected ducks and noninfected ducks. This analysis generated 65,536 unigenes. Of these, 6458 genes were found to be significantly differentially expressed between the infected and noninfected groups. The symptom and pathology of ducks infected with MDRV-YB was correlated with the greater proportion of decreased expression genes (4906) than increased expression (1552) level. Gene ontology analysis assigned differentially expressed genes to the categories: “biological processes,” “cellular functions,” and “molecular functions.” Differentially expressed genes involved in the innate immune system were analyzed further, and 128 of these genes showed decreased expression and 86 showed increased expression between the infected and noninfected groups. These genes represented the Janus kinase–signal transducer and activator of transcription signaling pathway, and the retinoic acid-inducible gene I (RIG-I)–like and Toll-like receptor (TLR) signaling pathways and included interferon (IFN) α, IFNγ, interleukin 6, RIG-I, and TLR4. The data were verified by SYBR fluorescence quantitative polymerase chain reaction (SYBR-qPCR). Our findings offer new insight into the host immune response to MDRV infection.