Three fungi of Pleurotus ostreatus Liuyuehui(PoL), Auriuolaria hispida Iwade(AhI) and Agrocybe aegirit(Aa) were inoculated with rice straw mixed substrate and fermented for 20 days. The collected rice straw substrate after the complete growth of hyphae was subject to the analysis of conventional nutrient composition, and the rumen chyme liquid from goat was also collected for characterization in vitro rumen fermentation for 72 hours to study the effects of different fungi fermentation treatments on the nutrient composition and in vitro rumen fermentation characteristics of rice straw substrate. The results showed the crude protein(CP) contents of PoL and AhI groups were significantly higher than that of Aa group, and the contents of neutral detergent fiber(NDF) and acid detergent fiber(ADF) in AhI and Aa groups were significantly lower than those of PoL groups. The methane, hydrogen and total gas production at 72 hours, dry matter degradation rate and total volatile fatty acid content of in vitro rumen fermentation in PoL group were significantly higher than those of the other two groups. In vitro rumen fermentation broth pH of PoL group was significantly lower than that of the other two groups. It was concluded that content of CP was higher in PoL rice straw treatment group, whose degradation degree of NDF and ADF were lower than those of culture materials, and in vitro rumen fermentation was better than the other two groups. PoL fermentation could be used to treat rice straw to improve its feeding value in practice.