This study investigated the effects of rumen-degradable starch (RDS) on lactation performance, gastrointestinal fermentation, and plasma metabolomics in dairy cows. Six mid-lactation cows, fitted with rumen, duodenum, and ileum cannulas, were used in a duplicated 3 × 3 Latin square design with 28-day periods. The cows were fed a low RDS (LRDS
62.18 %), medium-RDS (MRDS
71.25 %), or high-RDS (HRDS
80.32 %) diet. The results showed that cows fed HRDS had diet a lower milk fat content by 14.77 % (LRDS) and 11.73 % (MRDS), while increased somatic cell count compared to the LRDS and MRDS groups (34.42 and 29.38 %, respectively). Additionally, rumen fluid pH was decreased in the HRDS group than in the MRDS and LRDS groups (7.81 and 7.08 %, respectively), while microbial protein (MCP) concentration was higher in the MRDS group. The HRDS group had lower concentrations of total volatile fatty acids (TVFA) and acetate in the ileal digesta than the LRDS group. The HRDS diet decreased neutral detergent fibre (NDF) digestibility compared with LRDS and MRDS groups (7.68 and 8.50 %, respectively), and reduced plasma concentrations of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), while increasing plasma lipopolysaccharide (LPS) and serum amyloid-A (SAA) levels. Pathway analysis revealed that starch and sucrose metabolism, galactose metabolism, and carbohydrate digestion and absorption were upregulated in the MRDS and HRDS groups. The HRDS diet had a tendency to negatively affect linoleic acid metabolism, glycerophospholipid metabolism, and alpha-linolenic acid metabolism. These findings provide insights into optimising feed efficiency and milk quality by regulating RDS levels in dairy cow diets.