Objective：This study aims to investigate the relationship between serum 25-hydroxyvitamin［D25（OH）D］ concentration and hand-foot-and-mouth disease（HFMD），and to evaluate the clinical significance of 25（OH）D concentrations in children with HFMD. Methods：Total 126 children with HFMD were divided into mild HFMD group（84 cases）and severe HFMD group（42 cases）. Another 58 healthy children in the same period were selected as control group. Serum 25（OH）D concentrations were measured in all subjects，and all subjects were divided into 25（OH）D normal（≥75 nmol/L）group，25（OH）D insufficiency（≥50-75 nmol/L）group and 25（OH）D deficiency（< 50 nmol/L）group according to serum 25（OH）D concentration. Serum calcium，D-dimer，lactate dehydrogenase and creatine kinase-MB levels were also measured in children with HFMD. Enterovirus nucleic acid was detected and typed by RT-PCR. Results：Serum 25（OH）D concentrations were generally low in all groups. The average concentration of serum 25（OH）D in severe HFMD group［（50.2±10.2）nmol/L］ was significantly lower than that in healthy control group［（73.1±12.3）nmol/L］and mild HFMD group［（71.2±13.5）nmol/L］；EV71 infection rate in severe HFMD group was significantly higher than that in mild HFMD group. Logistic regression analysis showed that serum 25（OH）D concentration was an independent factor affecting the prognosis of HFMD children. Conclusion：Serum 25（OH）D concentration has clinical value in judging and predicting the progress of HFMD. Further evidence is needed to demonstrate the clinical value of 25（OH）D in diagnosis of HMFD.