The present study investigated the anti-diabetic effects of Oligostilbenes extracts (Olie) from Iris lactea Pall. var. chinensis (Fisch.) Koidz ( I. lactea ) and the potential mechanisms, in high-fat-diet (HFD)-induced diabetic mice and 3T3-L1 adipocytes. Olie are a group of major active extracts from I. lactea that have been used as nutraceutical because of their antioxidant activity. Six-week Olie treatment improved fasting blood glucose levels, as well as blood lipid profiles in HFD/streptozocin (STZ)-induced diabetic mice, compared with non-treated mice. Olie treatment upregulated the levels of phosphorylated of AMPK and lipolysis-related proteins, while the hepatic expression of ACC and FAS in diabetic mice was inhibited. In cultured 3T3-L1 cells, Olie (2−15 μg/mL) treatment dose-dependently suppressed the differentiation into mature adipocytes and lowered cellular lipid accumulation. Consistently, Olie reduced expression of adipogenic transcription factors including CCAAT/enhancer-binding protein β (C/EBPβ) and peroxisome proliferator-activated receptor γ (PPARγ). In addition, mitochondrial function in 3T3-L1 adipocytes was improved after Olie treatment. Taken together, our findings indicate that a lipid-lowering effect of Olie in HFD/STZ-induced diabetic mice and adipogenesis/ lipogenesis suppressing effect in 3T3-L1 cells, via regulating the expression of lipid metabolism-related proteins.