Objective To construct and validate a macrophage migration inhibitory factor (mif) gene knockout strain of Toxoplasma gondii RH strain. Methods Single guide RNA (sgRNA) and site-directed mutation primers the RH strain of T. gondii Tgmif gene were designed. The pSAG1::Cas9-U6::sgUPRT plasmid was mutated into the pSAG1::Cas9-U6::sgTgmif plasmid targeting the Tgmif gene. A Tgmif donor plasmid containing 3 fragments of 1 001 bp upstream of Tgmif (Tgmif-up), dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) and 1 011 bp downstream of Tgmif (Tgmif-down) was constructed. The Tgmif donor DNA was amplified from the Tgmif donor plasmid by PCR. The pSAG1::Cas9-U6::sgTgmif plasmid and Tgmif donor DNA were mixed and electroporated into the tachyzoites of wild-type T. gondii RH strain (RH_WT) and cultured with pyrimethamine for 7 days prior to screening to obtain the stable expression of pyrimethamine-resistant tachyzoites. The upper and lower homology arms of dhfr-ts and Tgmif were amplified by PCR to identify Tgmif knockout monoclonal strains (RH_ΔTgmif). The protein of RH_ΔTgmif and RH_WT tachyzoites was extracted and the expression of TgMIF protein was analyzed by Western blotting. RH_ΔTgmif was cultured in human foreskin fibroblasts (HFF) cells for 10 passages, and the number of tachyzoites per 100 HFF cells was counted by Giemsa staining to evaluate the proliferation of RH_ΔTgmif in HFF cells in vitro. RH_WT was used as the control. Twenty BABL/c mice were randomly divided into RH_WT group and RH_ΔTgmif group (10 mice/group). The mice were injected intraperitoneally with tachyzoites of RH_WT or RH_ΔTgmif strains (1 000/mouse) for each group respectively. The survival of mice was recorded. Independent sample t-test was used to compare the proliferation of T. gondii, and Log Rank test was used to compare the survival rate of mice. Results PCR results showed that the Tgmif donor DNA fragment containing 3 fragments, including Tgmif-up, dhfr-ts and Tgmif-down was 5 049 bp long, which was as expected; RH_ΔTgmif strain amplified dhfr-ts upstream and downstream homology arm bands of 1 387 bp and 1 524 bp, respectively, while RH_WT strain had no corresponding bands; a 1 837 bp Tgmif band was amplified in RH_WT strain, while no corresponding band in RH_ΔTgmif strain. The results of Western blotting showed that the RH_WT strain had a protein band at a relative molecular mass (M_r) of 12 500, while the RH_ΔTgmif strain had no corresponding protein band. The results of Giemsa staining showed that the tachyzoites of RH_ΔTgmif strain were (2 986 ± 69.20) per 100 HFF cells, which was higher than the RH_WT strain (2 067 ± 51.08) (t = 18.50, P < 0.01). The results of the in vivo virulence test showed that the mice in the RH_WT group started to die on the 7th day and all died on the 9th day; the mice in the RH_ΔTgmif group started to die on the 5th day and all died on the 7th day, and the difference between the two groups was statistically significant (χ² = 17.45, P < 0.01). Conclusion The Tgmif gene knockout strain RH_ΔTgmif was successfully constructed, and the RH_ΔTgmif strain of Toxoplasma was more virulent than the RH_WT strain.