ETHNOPHARMACOLOGICAL RELEVANCE: Gaultheria leucocarpa var. yunnanensis, a distinguished member of the Gaultheria Kalm ex L. in the Ericaceae family, has been traditionally employed in the southwestern regions of China for the efficacious treatment of rheumatoid arthritis (RA). The anti-RA fraction (ARF) derived from Gaultheria leucocarpa var. yunnanensis has been previously demonstrated to effectively alleviate RA in vivo and in vitro. AIM OF THE STUDY: This research endeavor is dedicated to surveying the pharmacokinetic (PK) processes of ARF within plasma and tissues, profiling its metabolites in vivo, discerning the material foundation of its therapeutic efficacy, and delineating its anti-RA mechanisms. MATERIALS AND METHODS: The prototype components and metabolites of ARF in plasma and seven tissues of RA rats were analyzed by LC-MS RESULTS: The quantity and diversity of identified prototypical components and metabolites of ARF in model rat plasma increased over time. The spleen exhibited the highest number of metabolites and prototypical compounds of ARF. The UPLC-QQQ-MS/MS and HPLC-DAD method were developed and validated for the quantification of three chemical markers in rat plasma and tissues, respectively. Three effective components (MSTG-B, MSTG-A, and Gautherin) demonstrated linear dynamics in plasma and tissues at an oral dosage of 3 g/kg ARF. The PK-PD models involving three components and four inflammatory cytokines aligned with the one company model, demonstrating a linear correlation through compartmental modeling and curve fitting analysis. Significant variations were identified in the concentrations of various amino acids and lipid metabolites among the CON, ARF, and MTX groups in comparison to the MOD group, which are intricately linked to the inflammation-immunity response. CONCLUSIONS: The three components displayed favorable bioavailability and were rapidly eliminated in RA rats, collectively exerting an anti-RA effect. The mechanism by which ARF mitigates RA is associated with the modulation of inflammation-immunity related metabolic pathways. The spleen may serve as the target tissue for ARF attenuating RA. These findings provide a robust foundation for rationalizing intervention strategies, elucidating biological mechanisms, and advancing the clinical application of ARF in the amelioration of RA.