This study evaluated the effects of sea buckthorn polysaccharides (SBP) on rumen fermentation in Hu sheep through in vitro fermentation. A single-factor randomized design was employed with four treatment groups in which SBP was added to a basal diet at 0, 1%, 2% and 3% of the total ration dry matter (DM). Based on gas production, degradation rates, and fermentation parameters, the control group and the 2% group were selected for 16S rDNA sequencing. The results showed that compared with the control group, the 2% SBP treatment significantly increased gas production rate (p < 0.05). Addition of 2% and 3% SBP significantly increased DM degradability (p < 0.05). SBP supplementation significantly increased fermentation fluid pH (p < 0.05) and altered volatile fatty acid profiles, increasing the acetate/propionate ratio as well as the concentrations of butyrate, isobutyrate and valerate. Compared with the control group, the addition of 2% SBP led to significant changes in the microbial composition of the in vitro rumen fermentation fluid. It significantly increased beneficial bacterial phyla and genera, such as Bacteroidetes, Rikenellaceae_RC9_gut_group and others, while reducing pathogenic bacteria (p < 0.05). Additionally, functional prediction revealed that the SBP group was significantly enriched in pathways related to carbohydrate metabolism, biosynthesis of amino acid, and biosynthesis of secondary metabolites (p < 0.05). In summary, adding 2% SBP to Hu sheep feed can improve the Rumen fermentation and microbial communities. However, higher doses did not yield additional benefits in these measured indicators.