The dysfunction of mesangial cells (MCs) in high-glucose (HG) conditions plays pivotal role in inducing glomerular sclerosis by causing the imbalance between generation and degradation of extracellular matrix (ECM) proteins, which ultimately leads to diabetic nephropathy. This study was designed to determine the function of forkhead box protein O1 (FoxO1), an important transcription factors in regulating cell metabolism and oxidative stress, in MCs in HG conditions. Up-regulation of fibronectin, collagen type IV, and plasminogen activator inhibitor (PAI-1) was observed under HG conditions in vivo and in vitro, accompanied with elevation of protein kinase B (Akt) phosphorylation and reduction of FoxO1 bioactivity. After overexpression of constitutively active (CA) FoxO1 in vivo and in vitro by using lentivirus vector, in vivo and in vitro, FoxO1 expression and activity was increased, in accordance with up-regulation of antioxidative genes (catalase and superoxide dismutase, leading to alleviated oxidative stress as well as attenuated Akt activity, whereas overexpression of wild type-FoxO1 only expressed partial effect. Moreover, CA-FoxO1 decreased the expression of fibronectin, collagen type IV, and PAI-1, causing amelioration of renal pathological changes and decrease of ECM protein deposition in glomerulus. Overexpression of CA-FoxO1 in renal cortex also decreased activin type-I receptor-like kinase-5 levels and increased signaling mothers against decapentaplegic (Smad) 7 levels, and simultaneously inhibited Smad3 phosphorylation. Results from in vitro study indicated that increased combination of FoxO1 and Smad3 may interfere with the function of Smad3, including Smad3 phosphorylation and translocation, interaction with cAMP response element binding protein (CREB)-binding protein, and binding with PAI-1 promoter. Together, our findings shed light on the novel function of FoxO1 in inhibiting ECM deposition, which is beneficial to ameliorate MC dysfunction.