The hypoglycemic effects of total flavonoids from Suillus luteus (SLFLA) were investigated in vitro and in vivo. The glucose consumption experiment in HepG2 cells showed that SLFLA had hypoglycemic activity in vitro. Moreover, SLFLA could improve insulin resistance and exhibited good hypoglycemic activity in the insulin resistance model. In streptozotocin-induced diabetic mice, SLFLA could effectively reduce blood sugar, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total cholesterol, triglyceride, alanine aminotransferase, and aspartate aminotransferase levels. Damage to the liver, kidney, and pancreas in diabetic mice was mitigated after intragastric administration of SLFLA. These results suggested that SLFLA might be a promising functional food or medicine for T2DM treatment.