The extraction and determination of the main active ingredient from the seeds of “Guiyou No.1”, a new variety of Shatian pomelo as the testing piece, were carried out. The antidiabetic effect of sodium glucose cotransporter 2 (SGLT-2) of the active ingredient, an important target of diabetes mellitus, was studied. The results showed that limonin, naringin and neohesperidin were the main active ingredient of “Guiyou No.1” seed. Among them, neohesperidin and naringin showed relatively good inhibitory effect on SGLT-2 activity, and showed hypoglycemic effect in diabetic mouse model, the hypoglycemic rate was 31.29% and 26.32%, respectively. The results are significant for the comprehensive development and utilization of “Guiyou No.1”.