Rumen bacteria can play an important role in the fatty acid deposition in the skeletal muscle of lambs. This study aims to explore the effects of arginine on carcass traits, meat quality, and rumen bacterial composition of lambs. The relationship between rumen bacteria and fatty acid deposition was also determined using metagenomic and gas chromatographs. Among them, metagenomic technology was used to analyze the changes in rumen bacteria after arginine supplementation in lambs. The gas chromatograph was to identify the fatty acid profile in the sheep muscle. The correlation between ruminal bacteria and muscle fatty acids was established after the experiment. Sixteen healthy lambs (three months old) with similar body weight were randomly divided into the control group (basal diet) and the arginine group (basal diet + 1% arginine). The feeding trial lasted for 90 days. The carcass traits were determined to calculate the related indexes after slaughter. Rumen contents were frozen and stored in liquid nitrogen at -80 ℃ for metagenomic analysis of rumen bacteria and volatile fatty acids. Samples were collected from the left longissimus dorsi muscle of the carcass, one part of which was used to determine meat quality, and another part was stored at -20 ℃ for the fatty acid composition. The results showed that there was a significant decrease in the backfat thickness of lambs in arginine (P<0.05), the lightness (L*), yellow (b*), and shear force of longissimus dorsi muscle (P<0.05), whereas, the redness (a*) value significantly increased (P<0.05), compared with the control group. Dietary arginine supplementation improved the color and tenderness of meat, indicating a positive effect on meat quality. The contents of pentadecanoic acid, heptadecanoic acid, and heptadecenoic acid in the arginine group were significantly lower than those in the control group (P<0.05). By contrast, the contents of tetracosenoic acid, α-linolenic acid, docosahexaenoic acid, and n-3 polyunsaturated fatty acids (PUFA) in the arginine group were significantly higher than those in the control group (P<0.05). The n-6/n-3 PUFA ratio in the arginine group decreased significantly (P<0.05). As such, the arginine enhanced the nutritional value of mutton more conducive to human health. Metagenomic analysis showed that the supplementation with arginine significantly increased the abundance of Clostridium, Selenomona, Ruminococcus, and Treponema in rumen (P<0.05). The abundance of Butyrivibrio, Parabacteroides, Succiniclasticum, Methanobrevibacter, and Butyrivibrio proteoclasticus decreased significantly (P<0.05). In addition, the content of butyric acid increased significantly (P<0.05) in the rumen of the arginine group. Dietary supplementation with arginine relatively affected the rumen bacterial community of lambs for the composition of bacterial metabolites. Arginine supplementation promoted the fermentation and degradation of rumen cellulose in lambs. Some key bacteria were significantly correlated with the fatty acid composition, among which Butyrivibrio shared a significant negative correlation with n-3 PUFA (P<0.05), while a significant positive correlation with the saturated fatty acids (P<0.05). Therefore, the rumen bacteria greatly contributed to the hydrogenation of fatty acids. In short, the abundance of hydrogenating bacteria was dominated in the presence of biohydrogenation, leading to the deposition of fatty acids in muscle. In addition, the correlation analysis between fatty acids and meat quality found that the content of fatty acids had a greater impact on the meat color and pH value. In summary, the arginine reduced the abundance of hydrogenating bacteria in the rumen and the occurrence of hydrogenation, thus promoting the deposition of beneficial fatty acids in meat for the high meat quality of lambs. These findings can also provide new nutritional intervention strategies for the high quality of mutton.