This study aimed to investigate the effect of fermented spent mushroom substrate fromPleurotus eryngii(SMPE) supplementation on production performance, meat quality and rumen bacterial community structure ofHusheep. 120 2-month-oldHusheep with average body weight [(13.50 ± 3.10) kg] were selected and randomly divided into 4 groups with 3 replicates per group and 10 sheep per replicate. The control group (RL1) was fed a total mixed ration (TMR), and group RL2, RL3 and RL4 were fed the basal diets supplemented with 15%, 30% and 45% fermented SMPE, respectively. The pretest period lasted for 10 days and the test period lasted for 150 days. The results showed that: (1) Difference (p< 0.05) was observed in average daily feed intake (ADFI) and feed conversion ratio (FCR) between RL2 and RL4 groups. The eye muscle area (EMA) and grade rule (GR) values in RL2 and RL3 were significantly higher than those in RL1 and RL4 groups (p< 0.05). (2) The contents of threonine, valerine, leucine, lysine, histidine, essential amino acids, flavor amino acids, aspartic acid, serine, glutamic acid and arginine of thelongissimus dorsimuscle in RL2 and RL3 groups were significantly higher than RL1 and RL4 (p< 0.05). (3) A total of 1,202,445 valid sequences were obtained from rumen ofHusheep fed different amounts of fermented feed, and the valid sequences were clustered into 9824 Operational Taxonomic Units (OTUs). (4) α diversity analysis showed that the richness and diversity of rumen bacterial communities inHusheep in RL1, RL2, RL3 and RL4 groups were significantly higher than RL0 (raw materials of fermented SMPE) group (p< 0.05).βdiversity analysis showed that the bacterial community structure was the most different between RL0 and RL3. (5) At the genus level, compared with RL1, the relative abundance ofChristensenellaceaeR-7 in RL3 group decreased significantly by 33.59%, the relative abundance ofPrevotellaceaeUCG001 in RL2, RL3 and RL4 decreased significantly by 50.41%, 62.24% and 49.17%, respectively, and the relative abundance ofRuminococcaceaeNK4A214 in RL2 group increased significantly by 35.01% (p< 0.05). In summary, the addition of fermented SMPE to TMR can significantly improve the production performance, meat quality and rumen bacterial community diversity and abundance ofHusheep.