Type 2 diabetes mellitus (T2DM) is a major global health issue. The development of T2DM is gradual and preceded by the pre-diabetes mellitus (pre-DM) stage, which often remains undiagnosed. This study aimed to identify novel pre-DM biomarkers in a high-fat diet (HFD)-induced pre-DM mouse model. Male C57BL/6J mice were fed either a chow diet or HFD for 12 weeks. Serum and liver samples were isolated in a time-dependent manner. Semi-quantitative assessment of secretory cytokines was performed by cytokine array analysis, and 13 cytokines were selected for further analysis based on the changes in expression levels in the pre-DM and T2DM stages. HFD-fed mice gained body weight and exhibited high serum lipid, liver enzyme, glucose, and insulin levels during the progression of pre-DM to T2DM. The mRNA expression of inflammatory and lipogenic genes was elevated in HFD-fed mice The mRNA expression of Fc receptor, IgG, low affinity Iib, lectin, galactose binding, soluble 1, vascular cell adhesion molecule 1, insulin-like growth factor binding protein 5, and growth arrest specific 6 was elevated in the pre-DM, which was confirmed by measuring protein levels. Our study identified novel pre-DM biomarkers that may help to delay or prevent the progression of T2DM.