Improving the nutrient utilization efficiency of ruminants is of utmost significance for both economic and environmental benefits. Optimizing dietary protein levels represents a key nutritional strategy to enhance ruminant growth performance and reduce nitrogen emissions. In a 63-day experiment, 24 healthy Hulunbuir lambs (initial weight 17.1 +/- 2.0 kg, 2.5 months old) were subjected to three treatments: a low-protein diet (LP; crude protein of 78.4 g/kg dry matter [DM]), a medium-protein diet (MP; crude protein of 112.0 g/kg DM), and a high-protein diet (HP; crude protein of 145.6 g/kg DM), with 8 lambs in each treatment (4 males and 4 females). Lambs in the MP treatment presented greater daily weight gain and feed conversion ratio than those in the HP treatment (P < 0.05, quadratically). Compared with the LP treatment, the MP treatment resulted in greater crude protein digestibility (P < 0.001, quadratically) and acid detergent fiber digestibility (P = 0.022, quadratically). In the serum, the urea nitrogen level increased quadratically with increasing dietary protein levels (P < 0.001), while the LP treatment exerted the highest concentrations of glutamate, glycine, alanine, and histidine (P < 0.05, quadratically). The ammonia nitrogen concentrations in the rumen and colon increased quadratically with increase in dietary protein levels (P < 0.05). The HP treatment increased the molar concentrations of isobutyrate and isovalerate in the rumen and colon (P < 0.05, quadratically). In contrast, the LP treatment decreased the molar proportion of acetate (P = 0.007, quadratically) and increased the molar proportion of butyrate (P < 0.001, quadratically) in the colon. The microbial diversity and structure were significantly altered by dietary protein level intervention across all gastrointestinal regions. The rumen of the MP treatment was enriched with fiber-degrading bacteria Fibrobacter_succeinogenes and starch-degrading bacteria Selenomonas_ruminantium. The colon in the LP treatment harbored microbial biomarkers including Escherichia spp. and Lactobacillus amylovorus, and the colon in the MP treatment was characterized by the enrichment of Solibacillus_cecembensis. These findings suggest that the MP diet with a crude protein content of 112.0 g/kg DM improved the growth performance and nutrient efficiency of lambs, which was achieved via the involvement of the gastrointestinal microbiota. (c) 2025 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).