Since the banning of antibiotics, the use of feed additives to improve meat quality to satisfy people's pursuit of high quality has become a research hotspot. Yeast culture (YC) is rich in proteins, mannan oligosaccharides, peptides, and yeast cell metabolites, etc., and its use as a feed additive has a positive impact on improving meat quality. So the study aimed to provide a theoretical basis for YC improving mutton flavor and quality by detecting and analyzing the effects of YC on muscle physicochemical properties, amino acids, fatty acids, flavor composition, expression of related genes, and rumen microbiota of lambs. A total of 20 crossbred F1 weaned lambs (Australian white sheep♂ × Hu sheep♀; average 23.38 ± 1.17 kg) were randomly assigned to 2 groups, the control group (CON) and the 1.0% YC supplemented group (YC) (n = 10), and were reared in separate pens. The experiment had a pre-feeding period of 10 d and a treatment period of 60 d. After the experiment, 6 lambs in each group were randomly selected for slaughtering. The results showed that dietary YC supplementation increased rumen total VFA and acetate concentrations (p < 0.05), and muscle carcass fat (GR), a∗ value, intramuscular fat (IMF), lysine (Lys), arginine (Arg), nonessential amino acid (NEAA), oleic acid (C18:1n9c), and eicosanoic acid (C20:1) contents were significantly increased (p < 0.05), while cooking loss and γ-linolenic acid (C18: 3n6) were decreased (p < 0.05). Furthermore, we found that dietary YC improved the types of flavor compounds, and the key flavor substances such as hexanal, nonanal, styrene, benzaldehyde, p-xylene, and 1-octen-3-ol contents were changed (p < 0.05). Additionally, the expression of fat metabolism related genes PPARγ, FASN, and FABP4 were increased. Adding 1% YC to lamb diets increased profits by 47.70 CNY per sheep after 60 d of fattening. All of which indicated that YC could improve meat quality, especially flavor, which may be related to the regulation of the relative abundance of rumen microorganisms Bacteroidota, Prevotella_7, Succiniclasticum and Lachnospiraceae_NK3A20_group.