Gestational diabetes (GD) is a pathological condition, affecting 2–5% of pregnant women. Diosgenin (DSG) possesses a variety of biological activities. The present study was designed to examine the effect of DSG on GD and to investigate the possible mechanism in C57BL/KsJ-Lep^db/+ (db/+) mice. We found that DSG could remarkably ameliorated GD in pregnant db/+ mice, as reflected by the improvement of glucose and insulin intolerance, and the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content. The results showed that DSG could inhibit oxidative stress in pregnant db/+ mice, as evidenced by decrease of thiobarbituric acid reactive substances (TBARS) content, increase of glutathione (GSH) level and superoxide dismutase (SOD) and catalase (CAT) activities. DSG could also attenuate the abnormal changes of lipid profiles, including TC, TG and LDL, in pregnant db/+ mice. The increase of the expression of sterol regulatory element-binding transcription factor-1 (SREBP-1) and its target genes, including fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl coenzyme A carboxylase (ACC), was inhibited by DSG in pregnant db/+ mice. Moreover, overexpression of SREBP-1 by LV-SREBP-1 injection could markedly inhibit the protective effect of DSG against disorder of glucose and lipid metabolism and oxidative stress in GD mice. The data demonstrated that SREBP-1 may be of major target of DSG that mediated its anti-diabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD.