Abstract: Objective To investigate the relationship between serum 25-hydroxyvitamin D3 〔25(OH)D3 〕 concentration and glucose metabolism. Methods This cross-sectional survey recruited 668 participants, and the demographic and clinical characteristics of the participants were obtained from questionnaires. Physical examination and blood biochemical examination were performed, then the participants were divided into three groups as normal glucose tolerant (NGT) group, impaired glucose regulation(IGR) group, and diabetes mellitus (DM) group. Results There was no statistical significant difference in serum 25(OH)D3 concentration among the three groups ( P>0.05). With comparison of different serum 25(OH) D3 concentration, there was no statistical significant difference in the incidence of DM and IGR, FPG, 2 h BG, HOMA-IR, ISI-composite, HOMA-β or ΔI30/ΔG30 ( P>0.05). Serum 25(OH)D3 concentration was not associated with DM, IGR, FPG, 2 h BG, HOMA-IR, ISI-composite, HOMA-β or ΔI30/ΔG30 ( P>0.05). Conclusion No relationship between serum 25(OH)D3 concentration and glucose metabolism is comfirmed.