Objective To analyze the gene expression profile in the A549 human lung adenocarcinoma cell line transfected with the expression vector of Toxoplasma gondii rhoptry protein 16Ⅲ (ROP16Ⅲ), using bioinformatics methods, in order to identify candidate pathogenic genes. Methods The eukaryotic expression vector pEGFP-N1-ROP16Ⅲ was constructed. The A549 cells were assigned to three groups: untransfected, pEGFP-N1 and pEGFP-N1-ROP16Ⅲ groups. Cells in the latter two groups were instantantaneously transfected with pEGFP-N1 and pEGFP-N1-ROP16 plasmid, respectively. After 48 h, the cells were harvested and RNA was extracted. After RNA purification, microarray hybridization analysis was performed to identified differentially expressed genes (DEGs) which were further analyzed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional enrichment classification and pathway analysis. ToppGene was used to screen for Toxoplasma ROP16Ⅲ pathogenic candidate genes from DEGs. The STRING online analysis software was used to analyze the protein-protein interaction (PPI) network for protein products of the candidate genes. Results A total of 566 genes were differentially expressed in A549 transfected with Toxoplasma ROP16Ⅲ, of which 382 genes were up-regulated and 184 genes down-regulated. Bioinformatics analysis of the DEGs revealed 997 GO annotations and 50 KEGG signaling pathways. The DEGs were significantly enriched in defense response, type I interferon signaling pathway, innate immune response, and response to external biotic stimulus. KEGG pathway enrichment analysis suggested the DEGs were most significantly enriched in antigen processing and presentation, cytosolic DNA-sensing pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway. In addition, 14 candidate genes were screened out from the DEGs by the ToppGene Suite. Among them, C-X-C motif chemokine 11 (CXCL11), toll-like receptor 3 (TLR3), C-C motif chemokine ligand 26 (CCL26), human leukocyte antigen E(HLA-E), promyelocytic leukemia protein (PML) and signal transducer and activator of transcription 2 (STAT2) had not been reported in Toxoplasma studies. The PPI network of the DEGs comprised 376 nodes and 1 152 edges, in which STAT2, HLA-E and PML were just located at the core position of the PPI network. Deletion of these key nodes led to the PPI network structure disorganized. Pathway enrichment analysis of 6 genes including CXCL11 resulted in 12 signaling pathways, which were mainly related to chemokine signaling pathway, Toll-like receptor signaling pathway, necroptosis, endocytosis and cytokine-cytokine receptor interaction. These bio-signal pathway may be related to the pathogenic mechanism of Toxoplasma ROP16Ⅲ. Conclusion CXCL11, TLR3, CCL26, HLA-E, PML and STAT2 were identified as candidate pathogenic genes, providing theoretical basis for further investigation of pathogenic mechanism of toxoplasmosis.