〔Abstract〕 Objective To investigate the effect and mechanism of phosphoprotein phosphatase 5 catalytic(PPP5C) on the migration, invasion and tumor stemness of human lung adenocarcinoma H1299 cells. Methods The PPP5C-pcDNA3.1 overexpression vector was constructed. PPP5C-pcDNA3.1 and pcDNA3.1 were transfected into H1299 cells, and H1299 stable cell lines were screened with G418. The mRNA and protein expression levels of PPP5C were identified by qRT-PCR and Western blot. The proliferation activity of H1299 cells was detected by drawing cell growth curve and cell clonal formation assay. The wound-healing assay and transwell assay were used to test the migration and invasion abilities of H1299 cells, respectively. The stemness of H1299 cells was evaluated by sphere formation assay. Results The PPP5C-pcDNA3.1 eukaryotic expression vector was successfully constructed and the expression levels of PPP5C significantly increased after transfection into H1299 cells. After overexpression of PPP5C in H1299 cells, the cell growth curve and clonal formation assay displayed that the proliferation ability was not affected, the migration and invasion of cells were significantly enhanced through wound-healing assay and transwell assay, accompanied by an increase in the expression of MMP9, stem cell spheroid assay showed a significant increase in stemness of cells, accompanied by increased expression of SOX2. Conclusion The proliferation ability of cells is not affected, the migration and invasion and the stemness of cells are enhanced by regulating MMP9 and SOX2 respectively, after overexpression of PPP5C in human lung adenocarcinoma H1299 cells.