In our previous study, diet directly impacted the microbiota of the rumen in twin lambs. The duodenum is the first part of the small intestine, so we seek to determine whether there is a difference in the digesta between the two feed groups HFLP (high fiber, low protein) and LFHP (low fiber, high protein), and its impact on the biodiversity and metabolism of the duodenum. Results showed that the number of Operational Taxonomic Units (OTUs) in the duodenum (2,373 OTUs) was more than those in the rumen (1,230 OTUs), and 143 OTUs were significantly different in the duodenum between the two groups. The two most predominant phyla were Bacteriodetes and Firmicutes, but this ratio was reversed between the rumen and duodenum of lambs fed different feedstuffs. The difference in the digesta that greatly changed the biodiversity of the rumen and duodenum could affect the microbial community in the gastrointestinal tract (GIT). Sixteen metabolites were significantly different in the duodenum between the two groups based on the metabolome analysis. The relationships were built between the microbiome and the metabolome based on the correlation analysis. Some metabolites have a potential role in influencing meat quality, which indicated that the diet could affect the microbiota community and finally change meat quality. This study could explain how the diet affects the rumen and duodenum’s microbiota, lay a theoretical basis for controlling feed intake, and determine the relationship between the duodenum’s microbiota and metabolism.