BackgroundDue to their diverse biological activities, polysaccharides derived from Tremella fuciformis have received growing attention. This study aimed to investigate the structural characterization of a purified polysaccharide (designated as PTP‐3a) derived from Tremella fuciformis and explore its interaction with gut microbiota in vitro.ResultsThe findings revealed that PTP‐3a had a molecular weight of 1.22 × 103 kDa and consisted of fucose, glucose, xylose, mannose, and glucuronic acid in a molar ratio of 0.271:0.016:0.275:0.400:0.038. The primary linkage types identified in PTP‐3a were 1,3‐linked‐manp, 1,4‐linked‐xylp, and 1,2,3‐linked‐fucp, with corresponding ratios of 0.215:0.161:0.15. In addition, PTP‐3a demonstrated notable thermal stability and exhibited a triple helical structure. Moreover, following in vitro fermentation for 48 h, PTP‐3a was efficiently utilized, resulting in a reduction in carbohydrate levels, the production of short‐chain fatty acids (SCFAs), and pH adjustment. Furthermore, during in vitro fecal microbial fermentation, PTP‐3a decreased the relative abundance of Firmicutes while increasing the proportions of Bacteroidetes and Proteobacteria, resulting in a significantly reduced Firmicutes/Bacteroidetes ratio. Additionally, PTP‐3a stimulated the growth of beneficial bacteria such as Parabacteroides merdae, Gordonibacter pamelaeae, Bifidobacterium pseudolongum, and Parabacteroides distasonis. Importantly, a strong correlation was observed between the production of SCFAs and specific microorganisms.ConclusionThese findings suggested that PTP‐3a held potential as a prebiotic for modulating the gut microbiota.This article is protected by copyright. All rights reserved.