Objective To explore the changes of ubiquitinated protein profile in host cells infected with Toxoplasma gondii. Methods After induced into macrophages, the human acute monocytic leukemia cell line (THP-1) cells were assigned into three groups: uninfected group, RH infected group, and ME49 infected group. Cells in the infection groups were infected with strain RH and strain ME49 T. gondii tachyzoites with difference virulence, respectively, for 4 h. Then total proteins were extracted from the cells, and the ubiquitinated proteins were enriched using anti-ubiquitin antibody FK-2, followed by protein separation by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative mass spectrometry identification. Human source information was searched from UniProt databse, and the protein species were identified using Mascot. The ubiquitination level of CDC42 was verified by Western blotting. The proteins were digested into peptides and underwent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using the DAVID Bioinformatics Resources 6.8, Gene Ontology (GO) annotation and enrichment analysis was performed for significantly differentially ubiquitinated proteins (DUPs) between the infection and control groups and between the infection groups with different virulence. The pathway enrichment of DUPs was analyzed with KEGG online tool. The protein-protein interaction (PPI) network was analyzed with online STRING 11.1. Results LC-MS/MS results showed that compared with the control group, there were 194 DUPs in the RH infected group, 47 DUPs in the ME49 infected group, and 31 DUPs in both groups. Western blotting assay confirmed that the ubiquitination level of host CDC42 was increased significantly after T. gondii infection. GO clustering analysis showed that the DUPs in the RH infected group were mainly involved in energy metabolism and organelles of protein synthesis and processing (67 proteins), while the DUPs in the ME49 infected group were mainly involved in cytoskeleton structure (11 proteins). With regard to biological process clustering, no significant difference was found between the RH and ME49 infected groups, however, the DUPs of the ME49 infected group were enriched in Ephrin receptor signaling pathway (3 proteins) and clathrin-mediated endocytosis proteins (2 proteins). With regard to molecular function clustering, the DUPs of the RH infected group were significantly enriched in GO terms of RNA transcription/processing (47 proteins), GTPase activity (7 proteins) and ubiquitin ligase (11 proteins), while the DUPs of the ME49 infected group were significantly enriched in actin filament binding (3 proteins) and other GO terms. The enrichment of KEGG signaling pathway showed that DUPs in the RH infected group were mainly enriched in ribosome (12 proteins), ubiquitin-mediated proteolysis (8 proteins), phagosome (7 proteins), nucleotide excision repair (4 proteins) and pathogenic E. coli infection (4 proteins); while the DUPs in the ME49 infected group were mainly enriched in ribosome (3 proteins), actin cytoskeleton regulation (3 proteins) and pathogenic E. coli infection (2 proteins). The main node proteins of DUP interaction network in RH infection group included 60S ribosomal protein L9, protein transporter SEC61 subunit α subtype 1 and RAS related C3 botulinum toxin substrate 1. And in the interaction network of ubiquitinated proteins shared by RH infection group and ME49 infection group, the main node proteins were a cluster of ribosomal proteins and cell division control protein 42. Conclusion T. gondii infection results in significant changes in the ubiquitinated protein spectrum of host cells, which is related to virulence of the parasite strain. The DUPs involve in cytoskeleton, ribosome and ubiquitin proteasome pathway.