Dietary crude protein reduction and supplementation with rumen-protected amino acids may improve nitrogen utilisation in sheep; however, their effects on different small-intestinal sites remain unknown. This study investigated the digesta microbiota in paired duodenal and jejunal samples from 38 growing male Hu sheep fed one of four experimental diets: a conventional protein control diet, a low-protein diet (LPD), an LPD supplemented with rumen-protected lysine (LPDL), and an LPD supplemented with rumen-protected methionine (LPDM). Overall, 76 paired digesta samples representing 38 matched duodenum-jejunum pairs were analysed using 16S rRNA gene sequencing, and PICRUSt2 was employed to predict microbial functional potential. Host-response parameters, including serum biochemical, antioxidant, and immune-related indicators, were analysed to provide additional physiological context. In both intestinal segments, dietary treatment had no effect on alpha diversity. Richness-based indices, community structure (Bray-Curtis), dominant taxa, and inferred functional potential differed between the duodenal and jejunal communities. The effect of intestinal segment was greater than that of dietary treatment, and taxonomic and predicted functional differences associated with diet were observed primarily in the jejunum. Serum catalase activity differed between treatments after false discovery rate correction, with lower activity in the LPDM group; however, this finding was considered contextual rather than mechanistic. Overall, intestinal segments showed a stronger association with the small-intestinal microbial pattern than dietary treatment, suggesting that the duodenal and jejunal microbiota should be investigated separately in ruminant nutritional trials.