Trehalose is a reducing disaccharide, acting as a protectant against various environmental stresses in numerous organisms. In plants, trehalose-6-phosphate synthase (TPS) plays a crucial role in trehalose biosynthesis.Anoectochilus roxburghii(Wall.) Lindl. is a prominent species of theAnoectochilusgenus, widely utilized as a health food. However, the functional analysis of TPS in this species has been limited. In this study, TPS genes were cloned fromA. roxburghii. TheArTPSgene, with an open reading frame spanning 2850 bp, encodes 950 amino acids. Comparative and bioinformatics analysis revealed that the homology was presented between the ArTPS protein and TPSs from other plant species. The ORF sequence was utilized to construct a prokaryotic expression vector, Pet28a-ArTPS, which was then transformed intoEscherichia coli. The resulting transformants displayed a significant increase in salt tolerance under the stress conditions of 300 mmol/L NaCl. Quantitative RT-PCR analysis demonstrated that the expression ofArTPSgenes responded to NaCl stress. The accumulation of G6P was upregulated, whereas the content of T6P exhibited an opposite expression trend. The glycometabolism products, including trehalose, exhibited notable changes under NaCl stress, although their variations may differ in response to stimulation. The content of kinsenoside, a characteristic product ofA. roxburghii, was significantly upregulated under NaCl stress. These results suggest that theArTPSgenes function in response to NaCl stimulation and play a key role in polysaccharide and glycoside metabolism inAnoectochilus. This study provides new insights into the engineering modification of the health foodA. roxburghiito enhance the medicinal activity of its ingredients.