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This study aimed to evaluate the effects of Pleurotus ostreatus spent mushroom substrate (P.SMS), which is characterized by high production but low utilization, on feed utilization and liver serum indices from the perspective of duodenal microorganisms. Forty-five 3-month-old Hu sheep were randomly assigned to five groups and fed diets in which whole-plant corn silage (WPCS) was substituted with P.SMS at levels of 0% (Con), 5% (PSMS5), 10% (PSMS10), 15% (PSMS15), or 20% (PSMS20). The results indicated that the addition of P.SMS complexly influenced the apparent digestibility of dry matter, organic matter, and crude protein, with PSMS10 showing the highest digestibility of these nutrients. P.SMS inclusion significantly affected serum alanine aminotransferase levels, with PSMS5 showing higher levels than both the Con and PSMS20 groups (p < 0.05). Importantly, the inclusion of P.SMS did not affect the richness and diversity of duodenal microorganisms. Significant differences in the phyla Verrucomicrobiota and Spirochaetota were observed between the Con and PSMS20 groups. The observed trend towards an increase in the genus Trichoderma (p = 0.057) suggests that P.SMS is susceptible to contamination by this genus, which in turn affects the structure of the intestinal flora. Furthermore, functional gene predictions indicated differences in amino acid metabolism among the groups (p < 0.05). In conclusion, feeding with 10% P.SMS resulted in the highest digestibility without adversely affecting the structure of the duodenal community or liver function.