Objective To characterize the function and localization of a putative protein TGGT1_310420 expressed on tachyzoite of Toxoplasma gondii. Methods The single guide RNA (sgRNA) targeting TGGT1_310420 was designed online. CRISPR/Cas9 construct targeting GGT1_310420 was obtained by mutating sgRNA of TgUPRT on pSAG1::CAS9-GFP-U6::sgUPRT. The knockout of TGGT1_310420 gene was performed through tagging the mCherry-Ty_HXGPRT sequence after the first 60 nt coding sequences of the endogenous copy using CRISPR/Cas9 genome-editing strategy. The correct insert was confirmed by PCR and DNA sequencing. The expressed fusion protein was detected by SDS-PAGE and Western blotting analysis, and its subcellular localization was determined by immunofluorescence microscopy. The phenotype of the knockout parasites was examined by a plaque assay. Results The CRISPR/Cas9 construct targeting TGGT1_310420 was successfully constructed and confirmed by sequencing. PCR analysis confirmed the integration of the mCherry-Ty_HXGPRT sequence into the correct locus and Western blotting assay detected a single protein band with an apparent relative molecular weight of 36 000. Immunofluorescent assay demonstrated that the first 20 amino acids of TGGT1_310420 fused to mCherry-Ty_HXGPRT was co-localized with T. gondii gliding associated protein 45(TgGAP45)to the parasite’s pellicle. Knockout of TGGT1_310420 gene revealed no measurable alteration in tachyzoites. Conclusion The first 20 amino acids of TGGT1_310420 are sufficient for pellicle targeting and the knockout of TGGT1_310420 did not change the phenotype of T. gomdii tachyzoite.