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Spexin plays a role in regulating glucose metabolism. This study investigated the spexin levels in different glycemic status and its association with insulin secretion in humans.A total of 462 subjects were recruited in this study, including 52 healthy subjects, 106 first-degree relatives (FDRs) of type 2 diabetes mellitus (T2DM), 115 impaired glucose regulation (IGR), 80 newly diagnosed T2DM, and 106 established T2DM. Serum spexin was measured using ELISA. The homeostasis model assessment of insulin resistance (HOMA2-IR) and β-cell function (HOMA2-β), and Stumvoll index estimating first- and second-phase insulin secretion were calculated.Spexin levels were higher in FDRs [235.53 pg/ml (185.28, 293.95)] and IGR [239.79 pg/ml (191.52, 301.69)], comparable in newly diagnosed T2DM [224.68 pg/ml (187.37, 279.74)], and lower in established T2DM [100.11 pg/ml (78.50, 137.34)], compared with healthy subjects [200.23 pg/ml (160.32, 275.65)]. Spexin levels were negatively correlated with fasting plasma glucose (FPG) (r = - 0.355, P 0.001), hemoglobin A1C (HbA1c) (r = - 0.379, P 0.001), and HOMA2-IR (r = - 0.225, P 0.001), and positively correlated with HOMA2-β (r = 0.245, P 0.001) after adjusting for age, sex, and BMI. Multivariate linear regression analysis showed that established T2DM and HOMA2-β were independently associated with serum spexin levels.Serum spexin levels represented as a bell-shaped curve along the glycemic continuum and is closely related with insulin secretion in humans.