Fermented shiitake mushrooms (Lentinus edodes) improved insulin sensitivity in both mother and offspring mice fed a high-fat diet. Supplementation with 1–5% fermented shiitake reduced serum insulin levels and HOMA-IR scores in pregnant mice, while also lowering fat deposition and improving lipid metabolism. Offspring from supplemented mothers showed reduced body fat and visceral fat, lower fasting glucose and insulin, and stronger activation of the liver PI3K/AKT signaling pathway. They also had higher litter weights and increased expression of fat-burning genes like adipose triglyceride lipase and hormone-sensitive lipase.