Objective To explore the correlation between serum 25-hydroxy vitamin D [25(OH)VitD] concentration and blood viscosity in type 2 diabetic (T2DM) patients. Methods Two hundred and thirteen T2DM patients were enrolled and the serum 25(OH)VitD concentration and whole blood viscosity at different shear rates (1/s, 5/s, 30/s, and 200/s) were measured with enzyme-linked immunosorbent assay and electronic blood rheology analyzer. Biochemical indexes such as plasma glucose and serum lipids were also measured. Patients were assigned to Q1 group (53 cases), Q2 group (54 cases), Q3 group (53 cases), and Q4 group (53 cases) according to the quartiles of serum 25(OH)VitD levels. The correlation between serum 25(OH)VitD concentration and blood viscosity at different shear rates was analyzed and a gender-stratified analysis was performed. Results The whole blood viscosity at different shear rates in Q4 group was significantly higher as compared with other three groups (P<0.0001) in T2DM patients, especially in female patients. The results of Spearman correlation analysis showed that serum 25(OH)VitD concentration was positively correlated to the whole blood viscosity at different shear rates (P<0.05). Multivariate stepwise regression analysis suggested that serum 25(OH)VitD concentration in female patients was independently correlated to the blood viscosity at different shear rates (1/s, 5/s, and 30/s) (P<0.05). Conclusion In T2DM patients, especially in female patients, serum 25(OH)VitD concentration is positively correlated to the blood viscosity. Clinical indexes such as blood viscosity and 25(OH)VitD should be comprehensively evaluated during the diagnosis and treatment of diabetes mellitus.