The tick Dermacentor silvarum is recognized as an emerging vector of marked medical and veterinary significance. The current study investigated the gene expression profiling of the unfed nymphal D. silvarum in response to low temperature using high-throughput transcriptome sequencing. A total of approximately 12-giga base pairs were produced, which yielded 87,500 unigenes, and among them, 29,815 unigenes were successfully annotated by all available databases (NCBI nonredundant, Swissprot, UniProtKB/TrEMBL, the clusters of orthologous groups databases, the Kyoto encyclopedia of genes and genomes database, and pfam database). After low temperature treatment (4°C for 10 days), 2,241 genes, including 1,160 up-regulated and 1,081 down-regulated genes, were differentially expressed. The differentially expressed genes were further enriched to GO terms and KEGG pathways, and our results showed that 133 genes were enriched in molecular function, 90 genes were enriched in cellular components, 107 genes were enriched in biological processes and 202 genes were assigned into different KEGG pathways. This study establishes a molecular basis for gene function analysis in the tick D. silvarum, and will provide theoretical information for mechanistic studies on the overwintering adaptations of ticks.