Objective To investigate the effect and its mechnism of ubiquitin ligase cullin3 (CUL3) on the proliferation, migration and invasion of gastric cancer cells. Methods The expression of CUL3 in gastric cancer and adjacent tissues and its relationship with clinical prognosis were analyzed based on TCGA database. HGC-27 and BGC-823 cells were transfected with CUL3 shRNA and empty lentivirus to construct a CUL3 knockdown gastric cancer cell line group (shCUL3 group) and an empty lentivirus transfection group (Vector group). CCK-8 assay, clone formation assay, EdU staining, scratch assay and Transwell assay were used to detect the changes of proliferation, migration and invasion ability of the two groups; Western blotting was used to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins and PI3K/Akt/GSK3β pathway proteins in two groups;The changes of migration and invasion ability of shCUL3 group were detected after application of PI3K agonist 740 Y-P. Results In the TCGA database, CUL3 was highly expressed in gastric cancer, and was significantly associated with poor prognosis of gastric cancer patients. Compared with Vector group, CCK-8, clone formation assay and EdU staining showed that the proliferation of gastric cancer cells in shCUL3 group was inhibited (P<0.05), and CUL3 shRNA could significantly inhibit cell migration and invasion (P<0.05); In addition, compared with Vector group, the expression of E-cadherin protein in shCUL3 group increased, and the protein levels of vimentin, β-catenin, p-PI3K, p-Akt and p-GSK-3β significantly decreased (P<0.05); In contrast, application of the PI3K agonist 740 Y-P could partially reverse the inhibitory effect of CUL3 shRNA on gastric cancer cell migration and invasion. Conclusions Knockdown of CUL3 can inhibit the proliferation, migration and invasion of gastric cancer cells, and the mechanism of action of CUL3 in gastric cancer may be related to the activation of PI3K/Akt/GSK3β signaling pathway.