Abstract: Objective To explore the interactions among H460, fetal lung fibroblast and mononuclear cells and the influence on the expression of MMP-2 and MMP-9 in three-dimensional culture model. Methods Four co-culture groups were designed in three-dimensional culture collagen gels, including H460 cells and fetal lung fibroblast groups, H460 cells and mononuclear cells groups, fetal lung fibroblast and mononuclear cells groups, and H460 cells and fetal lung fibroblast and mononuclear cells groups. The expressions of MMP-2 and MMP-9 were detected by gelatin zymography in condition medium. Results The activity and expression of proMMP-2 and MMP-2 were enhanced in co-culture groups of H460 cells and fetal lung fibroblast, and also enhanced in co-cultured fetal lung fibroblast with mononuclear cells. Co-cultures of H460 cells and mononuclear cells resulted in the production of proMMP-2 and MMP-9.When H460 cells co-cultured with fetal lung fibroblast and mononuclear cells,the activity and expression of MMP-2 and MMP-9 were significantly up-regulated. Conclusion The interactions among H460 cells,fetal lung fibroblast and mononuclear cells may promote lung cancer invasion and metastasis through Up-regulating MMP-2 and MMP-9 expression.