Simple SummaryIntestinal development is a gradual process that starts before birth and continues throughout the postnatal period, with nutrition playing a critical role. In this study, we performed transcriptome sequencing to investigate the effects of milk replacer (MR) feeding level on intestinal gene expression in lambs and its relationship with intestinal function and development. The intensive use of MR was observed to promote intestinal morphological development and digestive enzyme activities and considerably affect intestinal gene expression, which was still significant at 14 days postweaning. Further, the intensive use of MR affected the insulin sensitivity of the intestinal tissue and regulated nutrient distribution and metabolism by synchronizing the expression ofAHSG,IGFBP1,MGAT2,ITIH, andCYP2E1in the jejunal tissue of lambs.AbstractAlthough early feeding strategies influence intestinal development, the effects of milk replacer (MR) feeding level on intestinal structure and functional development and underlying regulatory mechanisms remain unclear. In this study, 14 male Hu lambs were fed MR at 2% or 4% of their average body weight and weaned at 35 days of age. The MR was produced by the Institute of Feed Research of the Chinese Academy of Agricultural Sciences, and it contains 96.91% dry matter, 23.22% protein, and 13.20% fat. Jejunal tissues were assessed by RNA-seq for differences in the gene expression of lambs at 49 days of age; regulatory pathways and mechanisms of the effects of early nutrition on intestinal function and development were analyzed, along with growth performance, feed intake, jejunal histomorphology, and digestive enzyme activities. Increasing MR- feeding levels increased dry matter intake and daily gain before weaning, as well as lactase, amylase, lipase, trypsin, and chymotrypsin activities and intestinal villus length and muscular thickness. Overall, 1179 differentially expressed genes were identified, which were enriched in nutrient metabolism, coagulation cascades, and other pathways. Further, intensive MR feeding affected insulin sensitivity to reduce excessive glucose interception by intestinal tissues to ensure adequate absorbed glucose release into the portal circulation and promoted lipid and protein degradation in intestinal tissues to meet the energy demand of intestinal cells by regulatingAHSG,IGFBP1,MGAT2,ITIH, andCYP2E1expression.