The diagnosis of short stature (SS) is of widespread importance for later treatment. In the present paper, a metabolomic method was used to analyze the metabolic characteristics of SS children caused by endocrine metabolic diseases in order to understand the underlying biochemical mechanism and provide a potential intervention strategy for SS. According to the clinical diagnosis and family investigation, all patients with SS were confirmed to be due to the endocrine disorders, especially GH deficiency (GHD). A nuclear magnetic resonance (NMR)-based metabolomic analysis of serum was used to identify the metabolic changes in 45 SS children from the 35 healthy controls (HCs). The disturbed metabolic network related to SS was correspondingly derived from the differential metabolites. The SS children demonstrated higher serum levels of citrate, phenylalanine, creatinine, and tyrosine and lower serum levels of glucose, serine, betaine, inositol, lysine, glycerol, and glutamine compared with the HCs. The results demonstrated that the disturbed glucose metabolism and metabolism and biosynthesis of amino acids are typical metabolic features of SS, and the lower levels of lysine and glutamine are the metabolic characterization of the affected growth axes and stress state of SS, respectively. The significant changes of those serum metabolites are able to be regarded as potential biomarkers for the diagnosis of SS. Accordingly, supplemental betaine in dietary pattern, the improvement of glycometabolism, and endogenous replenishment of lysine and glutamine allow the possible treatment strategy for SS.