This study aimed to study the roleof microRNA (miR)‐181b and its target TIMP3 in the development of diabetic nephropathy (DMN) via inhibiting the apoptosis of mesangial cells. Real‐time polymerase chain reaction (RT‐PCR) was adopted to compare the miR‐181b expression between subjects with diabetic nephropathy (DN) and normal control. In addition, luciferase assays were utilized to explore the regulatory relationship between TIMP3 and miR‐181b. Real‐time PCR and densitometry analysis were conducted to measure the levels of TIMP3 mRNA/protein in DMN or in cells treated by miR‐181b inhibitors, miR‐181b mimics, and TIMP3 siRNA. And the 3‐(4,5‐dimethythiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay was adopted to study the effect of miR‐181b on cell survival and apoptosis. miR‐181b expression was much higher in the DN group, and the results of computational analysis identified TIMP3 as a miR‐181b target. The luciferase activity of cells transfected with wild‐type TIMP3 and mutant2 TIMP3 was significantly reduced, whereas the luciferase activity of cells transfected with mutant1 TIMP3 was evidently higher. Furthermore, a negative regulatory relationship was established between TIMP3 and miR‐181b expression with a correlation efficient of −0.5351. The levels of TIMP3 mRNA/protein expression were apparently increased in the DN group. In addition, the treatment of cells with miR‐181b mimics and TIMP3 siRNA remarkably lowered the levels of TIMP3 mRNA/protein, whereas the transfection of cells with miR‐181b inhibitors notably elevated the expression of TIMP3 mRNA/protein. miR‐181b promoted the survival of cells and inhibited their apoptosis. The miR‐181b expression was related to the development of DMN and could be used as a prognosis biomarker of DMN in the patients with DM.