Abstract: Objective To investigate the effect and mechanism of up-regulating metalloproteinase tissue inhibitors (TIMP3) by targeted regulation of miR-21-5p to suppress matrix degradation (ECM) and epithelial mesenchymal transformation (EMT) in bladder cancer cells. Methods Bladder cancer SCSP-571 cells were cultured on 96-well plates and divided into Control group, Plasmid group, miR-21-5p silencing group, TIMP3 silencing group and miR-21-5p+TIMP3 silencing group. The cells in Control group were cultured as normal, cells in Plasmid group were co-cultured with plasma, cells in miR-21-5p silencing group were co-cultured with miR-21-5p inhibitor, cells in TIMP3 silencing group were co-cultured with TIMP3 -shRNA plasma, and cells in miR-21-5p+TIMP3 silencing group were co-cultured with both miR-21-5p inhibitor and TIMP3 -shRNA plasma. When cells were cultured for 72 h, the mRNA expression of miR-21-5p and TIMP3 in each group was determined by fluorescence quantitative PCR and the expression of ECM and EMT markers in each group was determined by WB experiment. Cell migration and invasion were measured by Transwell assay. Results Compared with Control group and Plasmid group, the expression levels of E cadherin and TIMP3 in miR-21-5p silencing group were increased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and the cell migration and invasion count were decreased. The expression levels of E cadherin and TIMP3 in TIMP3 silencing group were decreased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and cell migration and invasion were increased. The difference had statistical significance (P＜0.05). The indexes of miR-21-5p+TIMP3 silencing group were between miR-21-5p silencing group and TIMP3 silencing group, and the differences were statistically significant (P<0.05). Conclusion The targeted regulation of miR-21-5p could inhibit the invasion and metastasis of SCSP-571 cells, and the mechanism may be related to the improvement of TIMP3 expression, thereby inhibiting the MMPs mediated ECM and EMT.