Background: Literature data on nutritional status in short stature children are unfortunately still scarce. The aim of the study was to compare the serum levels of 25(OH)D2, 25(OH)D3, total 25(OH)D and whole blood trace elements between short stature and healthy controls in Chinese children.Methods: A case-control study including 370 short stature (SS) children and 398 healthy controls (HC) was performed in Mianyang Central Hospital from January 2017 to June 2020. Serum 25(OH)D2 and 25(OH)D3 were accurately measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were summed to obtain total 25(OH)D. Whole blood concentrations of calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe), manganese (Mn), selenium (Se) and lead (Pb) were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) method. Results: 25(OH)D2 and total 25(OH)D levels in the SS group were significantly lower than the HC subjects (both P<0.05). Besides, both 25(OH)D2 and 25(OH)D3 were positively correlated with total 25(OH)D in the SS and HC groups. 25(OH)D2 levels had weakly negative association with 25(OH)D3 among healthy subjects, whereas no correlation between 25(OH)D2 and 25(OH)D3 levels was found in short stature group. Otherwise, a significant elevation was observed in Zn (P<0.001), Fe (P<0.001), and Se (P=0.027) in SS patients, while a statistically significant decrease in Cu (P=0.002) and Mn (P<0.001) was found. Additionally, the significant correlation of serum levels of 25(OH)D2, 25(OH)D3, total 25(OH)D and trace elements were observed between SS and HC group.Conclusion: SS children had more severe deficiency of 25(OH)D2 and total 25(OH)D than healthy subjects. In addition, deficiency of Cu and Mn, but elevation of Zn, Fe and Se were observed. Achieved information about vitamin D and trace element status in SS subjects could further improve the nutritional status of these patient groups.