The effects of palm kernel oil-2 monounsaturated fatty acid (OPO) on short-chain fatty acid (SCFA) levels in different segments of mouse intestine and its underlying mechanisms were investigated. Male C57BL/6J mice were randomly divided into control, low-dose OPO (LOPO, mass fraction 2.25%), and high-dose OPO (HOPO, mass fraction 9.00%) groups. After 4 weeks of feeding, SCFA levels in different intestinal segments were measured, and metabolomics and transcriptomics analyses were conducted to evaluate the effects of OPO intervention on intestinal metabolic networks and gene expression. The results showed that OPO intervention significantly increased SCFA levels, particularly acetate, propionate, and butyrate, and mass fractions in the jejunum and colon, up to 115.59%, 273.71%, and 188.74%, respectively, under HOPO intervention. However, in the ileum, the acetate and butyrate mass fractions in the high-dose OPO group were significantly reduced to 39.26% and 44.79%, respectively. Metabolomic and transcriptomic analyses revealed that OPO intervention significantly affected pathways related to lipid metabolism, digestion and absorption, and signal transduction, with the "fat digestion and absorption" pathway being highly enriched. This study demonstrated that OPO intervention optimizes intestinal digestion and absorption processes, dose-dependently increases SCFA levels in different mouse intestinal regions (particularly the jejunum and colon), thereby providing a theoretical basis for intervention strategies promoting host metabolic health.