Green tea residues are the by-product of tea processing and they contain a large number of bioactive ingredients. Steam explosion has been recognized as one of the most innovative pretreatments for modifying the physicochemical characteristic of polysaccharides from lignocellulosic materials. However, the comparison of biological activity of steam exploded (SE-GTR) and unexploded (UN-GTR) green tea residue polysaccharides was still unclear, which prompted the determination of the efficacy of steam explosion in tea residue resource utilization. In this study, the effects of two extracted polysaccharides UN-GTR and SE-GTR on human gut microbiota in vitro fermentation were conducted. The results showed that after steam explosion pretreatment, SE-GTR displayed more loose and porous structures, resulting in higher polysaccharide content (2483.44±0.5 μg/mg) compared to UN-GTR (1903.56±2.6 μg/mg). In addition, after 24 h fermentation, gut microbiota produced more beneficial metabolites by SE-GTR. The largest SCFAs produced among samples was acetic acid, propionic acid and butyric acid. Furthermore, SE-GTR could regulate the composition and diversity of microbial community, increasing the abundance of beneficial bacteria, such as Bifidobacterium. These results revealed that steam explosion pretreatment could be a promising and efficient approach to enhance the antioxidant activity and bioavailability of polysaccharides isolated from tea residues.