Mycorrhizal symbiosis represents a ubiquitous mutualistic relationship in nature, wherein mycorrhizal fungi enhance the host plant’s ability to absorb water and nutrients from the soil. In return, the host plant supplies the fungi with essential nutrients necessary for their metabolic activities. However, research focusing on the regulatory mechanisms governing mycorrhizal symbiosis in Dendrobium officinale remains limited. This study systematically investigates the regulatory mechanisms of mycorrhizal symbiosis on transcriptional synthesis in D. officinale by establishing a mycorrhizal symbiotic system, complemented by phenotypic observation, physiological measurement, and transcriptome sequencing. The results indicate that mycorrhizal symbiosis promotes both growth and nutrient absorption in D. officinale, concurrently increasing polysaccharide content. Through transcriptome analysis, we identified 59 differentially expressed genes associated with polysaccharide metabolism, alongside key genes and transcription factors integral to the regulatory network. Notably, the glycosyltransferase gene DoUGT83A1 was found to negatively regulate the mycorrhizal symbiotic system when heterologously expressed in tomato. This study provides a fundamental theoretical basis for elucidating the molecular mechanisms underlying polysaccharide synthesis in D. officinale and offers new insights for optimizing cultivation practices to enhance medicinal quality.