Abstract: The objective of this study was to evaluate the effect of dietary probiotic supplementation on protein synthesis and flavor compounds in lambs. Two experimental groups were randomly allotted for the 18 three-month-old crossbred ewes of Dorper × Small-Tailed Han sheep. The control group was fed a basic diet, and the probiotic group was supplemented with Clostridium butyricum at 5 g/head on the basis of the control diet (the viable number of Clostridium butyricum was 5.0×108 CFU/g). The pre-feeding was set as 7 days, and the feeding period lasted for 90 days. After that, the selected lambs were transported into the abattoir for slaughter, where the longissimus dorsi muscle was immediately dissected and weighed after slaughter. One part of the muscle samples were stored at -80 ℃ for the determination of enzyme activity and gene expression that related to protein synthesis, and another part was stored at -20 ℃ for the determination of flavor substances. Muscle weight, flavor substances, muscle fiber morphology, enzyme activities, and the expression of protein synthesis genes were determined by gas chromatography mass spectrometry (GC-MS) and real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that compared with the control group, the weight of longissimus dorsi muscle in the probiotic group increased significantly (P<0.05), while the diameter and cross-sectional area of muscle fibers increased significantly (P<0.01), indicating that the probiotics were enhanced the muscle weight of lambs. The activity of branched chain aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKDH) significantly increased in the muscle of the probiotic group (P<0.05), indicating that the dietary supplementation with probiotics accelerated greatly the metabolism of branched-chain amino acids in lambs. The gene expressions of insulin-like growth factor 1 (IGF-1), protein kinase B (Akt1), mammalian target of rapamycin (mTOR), p70 ribosomal proteinS6 kinase (p70S6K), and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) in the probiotics group were higher than those in the control group (P<0.05), whereas, the gene expressions of forkhead box protein o3 (FoxO3) and muscle atrophy F-box (MAFbx) were significantly lower than those in the control group (P<0.05). There was no significant difference in the muscle ring-finger protein-1 (MuRF1) gene expression between the two groups (P>0.05). It was confirmed that the probiotics promoted protein synthesis, but inhibited the protein degradation of lambs, when increasing the gene expressions of Akt1, mTOR and p70S6K, and reducing the gene expressions of FoxO3 and MAFbx via the Akt signaling pathway. There was some influence of dietary probiotic supplementation on the composition and relative contents of flavor compounds in lambs. Compared with the control group, the content of hexanal, heptanal, nonanal, and decanal were significantly reduced in the probiotic group (P<0.05), whereas, the content of 1-octanol and 1-octen-3-ol increased significantly (P<0.05). Octanal, nonanal, (E)-2-nonenal, decanal, (E)-2-decenal, dodecanal, 1-octen-3-ol, and phenol greatly contributed to the formation of lamb flavor in the probiotic group, imparting fruit-like, onion-like, sweet, mushroom, grassy and rose-like flavors. Correlation analysis showed that there was a correlation between the critical flavor substances and protein synthesis-related indicators, in which the gene expression of IGF-1 was significantly negatively correlated with the (E)-2-nonenal, decanal and (E)-2-decenal (P<0.05), while positively correlated with the 1-octen-3-ol (P<0.05). The gene expression of Akt1 was negatively correlated with the (E)-2-nonenal (P<0.05). In conclusion, the addition of probiotics into the diet can be expected to improve the protein synthesis level of lambs through the Akt signaling pathway, which in turn affected the flavor of lamb. The finding can also provide a theoretical basis and reference to regulate the quality of lamb via the diet.