The gestational period is a critical developmental window where maternal nutrition programs offspring growth and long-term health. This study evaluated dietary rumen-protected glutamine (RP-Gln) supplementation during late gestation on lamb growth performance, and its regulatory effects on metabolic, immune, and microbial parameters in ewes and offspring. Compared with controls, moderate RP-Gln significantly improved lamb growth, increasing birth weight, 15-day body weight, and average daily gain (p < 0.01). In ewes, RP-Gln elevated serum GGT activity (p < 0.05), improved lipid metabolism, and reduced offspring muscular stress. Antioxidant capacity was enhanced in both ewes and lambs, with higher antioxidant enzyme activities and lower MDA levels (p < 0.05). RP-Gln also remodeled gut microbiota by promoting beneficial genera like Lactobacillus and Faecalibacterium, and reinforcing dam–offspring microbial transmission. Metabolomics revealed elevated polyunsaturated fatty acids, vitamin C, and tyrosine derivatives in ewes, and enhanced aromatic amino acid biosynthesis in lambs. In summary, glutamine supplementation in late-gestation ewes enhances maternal antioxidant and anti-inflammatory capacity, optimizes gut microbiota, and stimulates n-3 PUFA synthesis. These benefits are transmitted to the fetus, improving offspring antioxidant function, immune regulation, and intestinal health. Glutamine also promotes aromatic amino acid synthesis while inhibiting the tryptophan–kynurenine pathway, collectively supporting lamb growth and health.