Objective To investigate the changes in the gene expression profile of human monocyte THP-1 cells overexpressing Toxoplasma gondii rhoptry protein 16 (ROP16) using transcriptome sequencing, and to screen immune response-related driver genes. Methods Human monocytic THP-1 cells were seeded onto 96-well plates. Cells in the overexpression group was transfected with ROP16 overexpression lentivirus for 72 h, and cells in the control group were treated with an equal volume of culture medium. The efficiency of cell transfection was checked under a fluorescence microscope. Total RNA was extracted from THP-1 cells and THP-1 cells stably expressing ROP16 using the TRIzol reagent, followed by transcriptome sequencing. Differentially expressed genes (DEG) were screened, and a volcano plot was generated. DEGs were subjected to cluster analysis, Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway enrichment analysis, and gene ontology (GO) functional enrichment classification to screen immune response-related driver genes in human monocytic THP-1 cells following T. gondii infections, and gene expression was quantified using qPCR assay. Differences of means between groups were tested for statistical significance with independent sample t-test. Results Fluorescence microscopy displayed green fluorescence in more than 90% of the cells in the overexpression group in each field of view, and no green fluorescence was observed in the control group. qPCR assay quantified a higher relative expression level of ROP16 mRNA in the overexpression group (1 083.484 ± 68.990) than in the control group (1.000 ± 0) (t = 22.9, P < 0.01), indicating the successful generation of THP-1 cells that stably expressed ROP16. A total of 312 DEGs were identified in THP-1 cells stably expressing ROP16, including 193 upregulated genes and 119 downregulated genes. KEGG annotations showed that the highest proportion of DEGs were annotated to the organism system (24.2%), with 85 items significantly enriched, among which 47 genes were significantly enriched in the immune system. KEGG enrichment analysis showed that DEGs were significantly enriched in 20 signaling pathways, and 6, 7, and 8 DEG were significantly enriched in three pathways related to the immune system, including Th1 and Th2 cell differentiation, natural killer cell-mediated cytotoxicity, and IL-17 signaling pathways, respectively. GO functional annotations showed that a total of 309 DEGs were annotated to 55 secondary node classifications under three primary node classifications of biological processes, cellular components, and molecular functions. GO enrichment analysis showed that DEGs were significantly enriched in inflammatory response, negative regulation of tumor necrosis factor products, cytokine production, positive regulation of interferon-γ production, and immune response-related pathways. RT-qPCR assay detected that higher relative expression of MAN2B1, FOS, C1QA mRNA (25.994 ± 0.382、60.584 ± 2.968、 36.759 ± 0.180) in cells in the ROP16 overexpression group than in the control group (1.000 ± 0.039、1.000 ± 0.015、1.000 ± 0) (t = 92.00, 28.39, 280.7, P < 0.05 or 0.01), and lower relative expression of LMNB1, IL-6, and IL-12 mRNA (0.728 ± 0.054, 0.517 ± 0.073, 0.587 ± 0.015) in the overexpression group than in the control group (1.052 ± 0.027、1.000 ± 0.039、1.000 ± 0.010) (t = 7.64, 8.24, 33.62, P < 0.05 or 0.01). Conclusion The gene expression profile of human monocyte THP-1 cells changes significantly and may play important roles in the immune response following T. gondii infection.