Objective To evaluate the influence on the invasion ability of T24 bladder cancer cell lines after silencing of NANOG gene. Methods Human urinary bladder cancer cells T24 were cultured and transfected with short hairpin RNA (shRNA) to silence NANOG gene. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA) were performed to detect the expression level of matrix metalloproteinase (MMP)-2, MMP-9 and inhibitor of matrix metalloproteinases-1 (TIMP-1). Wound-healing assay and invasion test were performed to determine the invasion capability of T24 cell lines. Results When NANOG gene was silenced, the expression of MMP-2 and MMP-9 was down-regulated (for MMP-2 by 63.21%, P<0.05; for MMP-9 by 73.55%, P<0.05). ELISA showed that the expression of MMP-9 in supernatant of T24 cells was down-regulated by 21.07% (P<0.05). Scratch test and invasive test showed that the invasive ability of cells decreased after NANOG gene was silenced (all P<0.05). Conclusion NANOG may modulate the invasion of bladder cancer cells through regulating the expression of MMP-2 and MMP-9. Key words: Bladder cancer; Metal matrix proteinase 2; Metal matrix proteinase 9; Short hairpin RNA; Invasion