ObjectiveThe objectives of this study were to evaluate the effects of different forage proportions in the fermented total mixed ration (FTMR) on growth performance, muscle fatty acid profile, and rumen microbiota of lambs.MethodsThirty 6-month-old small tail Han sheep × Ujumqin lambs with initial body weight (BW) of 27.8 ± 0.90 kg were selected for the test and divided into two groups of 15 sheep in each treatment (three pens per treatment and five lambs per pen) according to the principle of homogeneity. Two isoenergetic and isonitrogenous diets were formulated according to the NRC. The diet treatments were designed as (1) OH treatment containing 25% alfalfa hay and 35% oat hay, and (2) AH treatment containing 35% alfalfa hay with 25% oat hay. The forage-to-concentrate ratio for both diets was 65: 35 (DM basis). Three replicates were randomly selected from each treatment to determine growth performance, fatty acid profile and rumen bacterial communities in lambs.ResultsResults revealed no statistically significant (p > 0.05) differences in dry matter intake and average daily gain between the two diet groups. Cholesterol and intramuscular fat were significantly (p > 0.05) higher in the AH group, while no statistically significant difference (p > 0.05) was found in pH24 value. The muscle fatty acid compositions of lambs were obviously (p < 0.05) influenced by the diet treatments. Compared with the OH group, the C16:1, C17:0, and C20:3n6 contents were higher (p < 0.05) in the AH group, whereas the content of C18:1n9c, C20:1, C18:3n3, and C22:6n3 was obviously (p < 0.05) increased in the OH group. The monounsaturated fatty acid (MUFA) contents were significantly higher in the OH group, whereas no significant differences (p > 0.05) were detected in saturated fatty acid (SFA) and polyunsaturated fatty acid (PUFA) contents among the two diet treatments. Bacterial composition was generally separated into two clusters based on principal coordinate analysis, and the OH group had a higher Shannon index. The relative abundance at the genes level of the Rikenellaceae_RC9_gut_group was obviously (p < 0.05) increased in the AH group and the relative abundances of Prevotella_1, Fibrobacter, and Bacteroidales_UCG_001_unclassified were obviously (p < 0.05) enriched in the OH group. Integrated correlation analysis also underscored a possible link between the muscle fatty acid compositions and significantly altered rumen microbiota.ConclusionOverall, oat-based roughage in FTMR could promote a beneficial lipid pattern in the Longissimus lumborum muscles of lambs. These findings provide a potential insight into diet effects on fatty acid profile and the rumen microbiome of lambs, which may help make decisions regarding feeding.