Objective：To study the inhibitory effect of supernatant from contact co-culture of human embryonic stem cells（hESCs） and tumor A549 cells on proliferation，apoptosis，invasion and migration of A549 cells. Methods：The direct co-culture system of hESCs H9 and lung cancer A549 cells was established，and the supernatant was tested in the inhibition of A549 cells. The A549 cell super－natant was used as the blank control group，and the hESC H9 supernatant was used as the control group. The inhibitory effects were examined in tumor cell morphology using microscope，cell proliferation with CCK-8 assay，and cell apoptosis with the Hoechst staining. Wound healing assay was used to detect the migration and invasion of tumor cells. Results：The number of tumor cells in the experimental group was gradually reduced and even apoptosis was observed under the microscope. CCK-8 method detected that the proliferation of tumor cells in the experimental group was significantly inhibited（P<0.05）. Hoechst staining found that the nuclei of the experimental group showed typical apoptotic morphology. The results of wound healing assay showed that the percentage of wound closure of tumor cells in the experimental group was significantly lower than that of the blank control group and the control group（P<0.05）. Conclusion：We conclude that the supernatant of co-culture cells had an inhibitory effect on tumor cells in vitro.