Background: Intramuscular fat (IMF) is a key determinant of meat sensory quality, but the effects of rumen-protected betaine (RPB) on IMF deposition in Tibetan sheep remain unclear. Methods: Sixty male, 3-month-old Tibetan lambs (Initial weight 17.72 ± 1.36 kg) were randomly assigned to a control (basal diet, n = 30) or RPB supplementation (basal diet supplemented with 0.08% RPB, n = 30) for 90 d. Results: Transcriptomic and lipidomic analyses revealed that RPB modulated glycerophospholipid metabolism-related genes (DGKI and AGPAT1) and altered lipid profiles, including PC (16:0/20:5) and PC (16:0/16:0). RPB increased T-AOC and SOD activities and decreased MDA content (p < 0.05). Intramuscular adipocyte area and diameter increased, while density decreased (p < 0.05). LC-MS/MS analysis showed that unsaturated fatty acids C14:1, C17:1, and C22:4 were significantly increased (p < 0.05). Muscle fiber density in the longissimus dorsi increased, whereas shear force, cooking loss, hardness, and chewiness were reduced (p < 0.05). MYH1, MYH2, and MYH7 expression were upregulated by 2.05-, 1.89-, and 2.73-fold, respectively. Conclusions: In summary, dietary RPB supplementation was associated with increased IMF deposition, which may contribute to improved meat quality, potentially through modulation of glycerophospholipid metabolism.