Abstract: 【Objective】 This study aimed to systematically elucidate the molecular response mechanisms of Anoectochilus roxburghii to low temperature stress through analysis of transcriptome and metabolome， providing theoretical basis and molecular targets for breeding cold-resistant varieties and innovating cultivation techniques. 【Method】 The in vitro plantlets of Anoectochilus roxburghii cultivated at normal temperature （25 ℃） used as the control （CK） were treated with low temperatures of 15 and 5 ℃ for 72 h. Transcriptome sequencing was employed to identify metabolic pathways and differentially expressed genes （DEGs） associated with cold damage. The effects of low temperature stress on metabolite biosynthesis were investigated using technologies of widely targeted metabolome and carbohydrate-targeted metabolome； the integrated transcriptomic and metabolomic analysis of carbon metabolism pathways was conducted using two-way orthogonal partial least squares （O2PLS） to identify key genes responding to low temperature stress. 【Result】 Under low temperature stress conditions， the pathways of metabolism， genetic information processing， environmental signal processing， cellular processes， and organismal systems of Anoectochilus roxburghii were markedly affected， while the photosystem Ⅱ light-harvesting complex proteins were markedly inhibited. The relative contents of four major classes of metabolites （carbohydrates and the derivatives， flavonoids， terpenoids， and organoheterocyclic compounds） increased significantly after low temperature stresses （P<0.05， the same below）， with the pronounced accumulation observed in trehalose， maltose， melibiose， lactose， and cellobiose. Conversely， the relative contents of alkaloids and their derivatives， plant hormones，and lipids decreased significantly after low temperature stresses； the content of the medicinal component kinsenoside decreased with declining temperature， while the contents of quercetin and its derivatives were significantly higher under the 15 ℃ treatment compared to other groups. In addition， carbohydrate metabolites such as trehalose， maltose， and melibiose showed a strong association trend in the O2PLS model with the gene involved in oxidative stress response （GSTU25）， mitochondrial energy metabolism-related genes （MT-CYB， MT-ATP6， MT-ND1， MT-ND4， MT-CO3， MT-CO1）， and the secondary metabolism-related gene （AAMT1）. 【Conclusion】 Low temperature stresses not only disrupt the basal metabolism of Anoectochilus roxburghii， but also significantly interferes with its secondary metabolic processes. To address low temperature stresses， a large number of genes related to environmental adaptation are activated to participate in the regulatory network， playing a pivotal role in this adaptive process. Moreover， moderate low temperature stresses can be seen as a potential stress elicitor to specifically increase the contents of the high-value medicinal components in Anoectochilus roxburghii.