Aims: Renal interstitial fibrosis and glomerulosclerosis are the characteristic presentation of diabetic nephropathy progression. Twist-1 overexpression contributes to renal fibrosis. Previous studies have demonstrated that pioglitazone (PIO), a PPAR-gamma agonists, can ameliorate renal fibrosis and protect renal function. However, whether PIO attenuates renal fibrosis and delays diabetic nephropathy progression by regulating Twist-1 expression remains unclear. Methods: Male Zucker diabetic fatty (ZDF) rats were randomly divided into 3 groups: (1) ZDF group, (2) ZDF + PIO group treated with PIO for 10 weeks, (3) ZDF + PIO + GW9662 group treated with GW9662 (a PPAR-gamma antagonist) and PIO for 10 weeks. Age-matched Zucker lean rats (ZL group) were used as a control group. Urinary albumin/creatinine ratio (UACR) and renal blood flow were measured. Renal histopathology and Twist-1 expression were determined by immunohistochemistry. The protein and mRNA levels of Twist-1 and PPAR-gamma were analyzed by Western blot and qRT-PCR. Results: PIO considerably reduced UACR and improved renal blood flow. This was associated with amelioration of glomerulosclerosis and tubulointerstitial fibrosis evidenced by the expression decrease of collagen I, aquaporin 1, alpha-SMA, transforming growth factor beta 1 and nephrin, although glycaemia remained high. Moreover, Twist-1 protein and mRNA expression in kidney of ZDF rats were significantly increased compared with ZL rats and PIO significantly decreased Twist-1 levels. Conclusions: This study shows that PIO can downregulate Twist-1 expression in the kidney, inhibit renal fibrosis and protect renal function in ZDF rats. These PIO-mediated effects are independent of glycemic control.