Objective To generate the Toxoplasma gondii sub3 (Tgsub3) gene knockout strain using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, investigate the in-vitro phenotypes of the Tgsub3 gene knockout strain, and examine the effect of Tgsub3 gene on adhesion, invasion and proliferation of T. gondii. Methods SgUPRT on the pSAG1::CAS9U6::SgUPRT plasmid was mutated to single guide RNA (sgRNA) using site mutation, and the pSAG1::CAS9-U6::sgSUB3 plasmid with the sub3 gene knockout was generated. The DHFR resistant donor fragments containing 40 bp upstream and downstream homology arms of the sub3 gene were amplified, and the sub3 gene knockout plasmid and donor fragments were co-transfected into T. gondii by electroporation. Following resistance selection by pyrimethamine and monoclonal screening, the sub3 gene knockout strain RH∆ku80∆sub3 (∆sub3) was identified using PCR assay. The in-vitro phenotypes of the ∆sub3 strain were analyzed with plaque, invasion, and proliferation assays. Using RH∆ku80 strain as a control, all statistical analyses were conducted using the software GraphPad Prism 9. Results PCR assay identified bands with expected sizes in the ∆sub3 strain, indicating successful generation of the ∆sub3 strain. Plaque assay showed that the sizes of plaques formed by RH∆ku80 and ∆sub3 strains were (60.42 ± 23.20) au and (2.21 ± 1.89) arbitrary unit, respectively (t = 17.79, P < 0.01), and invasion assay showed that the invasion efficiencies of RH∆ku80 and ∆sub3 strains were (37.94 ± 18.18) % and (22.97 ± 15.36) %, respectively (t = 0.89, P > 0.05). Proliferation assay showed that the proportions of parasitophorous vacuoles containing 8 and more tachyzoites of RH∆ku80 and ∆sub3 strainswere (56.33 ± 8.58) % and (39.67 ± 11.84) %, respectively, and the proportions of parasitophorous vacuoles containing 4 and fewer tachyzoites of RH∆ku80 and ∆sub3 strains were (43.67 ± 8.58) % and (60.33 ± 11.84) %, respectively. Compared with control strain, the number of tachyzoites within the parasitophorous vacuole was significantly decreased (F = 17.93, P < 0.01). Conclusion The ∆sub3 gene knockout strain is successfully generatedand absence of the Tgsub3 gene affects the growth and reproduction of T. gondii tachyzoites.