[Objective] In this study, we used high throughput sequencing (RNA-Seq) to investigate the gene expression profiles in human monocyte-derived macrophages (THP-1) before and post-infection of vaccinia virus (VACV).[Methods] We analyzed differentially expressed genes in human macrophages with and without vaccinia virus infection by RNA-seq and performed the KEGG, GO, and STRING network analysis to study the immune-related signal alterations during infection.[Results] A total of 4796 genes were differentially expressed (2416 genes upregulated and 2380 genes downregulated) of VACV treatment THP-1 versus control. Further KEGG enrichment analysis shows that differential genes expression in VACV treatment THP-1 cells were mainly involved in metabolism, signal transduction, immune system, infectious diseases and other pathways. Gene Ontology (GO) functional annotation indicated that these genes were mainly enriched on cell function regulation, metabolism, immune regulation and other life processes. The STRING online database analysis showed that JUN, CHUK, IL1B, PYCARD protein were higher in the infected THP-1 cells. [Conclusion] Vaccinia virus can induce the differential expression of various genes in host cells, and multiple biological processes. We conducted in-depth analysis of immune-related signal pathways, and found the C-type lectin receptor-, NOD-like receptor-, and Toll-like receptor- signaling pathway are involved in inflammation responses induced by vaccinia virus infection. These results provide new insights not only in exploring the mechanism of interaction between vaccinia virus and host, but also in applying it in treatments of infectious diseases and cancers.