Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by glucose metabolic disorders, and gluconeogenesis inhibiting is a promisingly therapeutic strategy for T2DM. Glucocorticoid receptor (GR) is tightly implicated in the regulation of gluconeogenesis, although the underlying mechanism remains obscure. Here, we discovered that small molecule, 5-chloro-N-[4-chloro-3-(trifluoromethyl)phenyl]thiophene-2-sulfonamide (FX5) as a new non-steroidal GR antagonist efficiently ameliorated glucose homeostasis in db/db and HFD/STZ-induced T2DM mice. The mechanism underlying the suppression of FX5 against gluconeogenesis was highly investigated. FX5 suppressed gluconeogenetic genes G6Pase and PEPCK in mouse primary hepatocytes and liver tissues of T2DM mice. Results of mammalian one-hybrid and transactivation as well as nuclear translocation assays totally evaluated the antagonistic features of FX5 against GR. Moreover, siRNA and overexpression related assays verified that FX5 alleviated gluconeogenesis either directly by antagonizing GR or indirectly through GR/HNF4α/miR122-5p signaling pathway. Our work has presented a new mode for GR antagonist in the regulation of gluconeogenesis, which is expected to highlight the potential of FX5 in the treatment of T2DM.