Objective To investigate the effect of costunolide on the malignant phenotype of gastric cancer cells and its possible mechanism.Methods Gastric cancer cells HGC-27 were cultured in vitro, and treated with costunolide at different doses (2.5, 10, 40 μmol·L^-1) for 24 h, or transfected with circ_0000285 small interfering RNA and then cultured for 24 h. The 40 μmol·L^-1 costunolide was used to intervene the HGC-27 cells transfected with circ_0000285 overexpression vector for 24 h. CCK-8 method and clone formation test were used to detect the cell proliferation. The flow cytometry was used to detect cell apoptosis. Scratch test and Transwell were used to detect cell migration and invasion. Western blotting was used to detect the protein expressions of Bcl-2, Bax, E-cadherin and N-cadherin in the cells, and RT-qPCR was used to detect the expressions of circ_0000285 and miR-409-3p. The dual luciferase reporter gene experiment was used to verify the regulatory relationship between circ_0000285 and miR-409-3p.Results Costunolide could reduce the OD value of HGC-27cells, the number of colonies formed, the migration distance, the number of invasion, and the protein expression levels of Bcl-2 and N-cadherin, while it could increase the cell apoptosis rate, and the protein expression levels of Bax and E-cadherin in a dose-dependent way. Costunolide could inhibit the expression of circ_0000285, but promote the expression of miR-409-3p in HGC-27cells. Interference with circ_0000285 expression could decrease the proliferation and migration of HGC-27 cells, and promote cell apoptosis. circ_0000285 could target and negatively regulate miR-409-3p. Overexpression of circ_0000285 could reverse the inhibitory effect of costunolide on HGC-27 cell phenotypes.Conclusion Costunolide may inhibit the malignant biological behavior of gastric cancer cells by regulating the circ_0000285/miR-409-3p axis.