Studies have shown that epoxyeicosatrienoic acids (EETs) can regulate glucose homeostasis, but the specific mechanisms need further exploration. The sodium-glucose co-transporter 2 (SGLT2) is highly expressed in diabetic kidneys, which further promotes renal reabsorption of glucose to respond to the hyperglycemic state of diabetes. Herein, whether EETs can be a latent inhibitor of SGLT2 to regulate glucose homeostasis in diabetic state needs to be elucidated. Our study demonstrated that EETs attenuated the glucose reabsorption via renal tubular epithelial cells in diabetic mice, which partly accounted for the beneficial effects of EETs on glucose homeostasis. Moreover, 14,15-EET suppressed SGLT2 expression in both diabetic kidney and renal tubular epithelial cells. Further, inhibition of NF-kappa B with BAY 11-7082 decreased insulin-induced SGLT2 expression while NF-kappa B overexpression reversed the above effects. In addition, 14,15-EET attenuated SGLT2 expression via inactivating NF-kappa B. Mechanistically, 14,15-EET attenuated NF-kappa B mediated SGLT2 transcription at the -1821/1812 P65-binding site. These results showed that EETs ameliorated glucose homeostasis via preventing NF-kappa B-mediated transcription of SGLT2 in renal tubular epithelial cells, providing a unique therapeutic strategy for insulin resistance and diabetes.