The dried rhizome of Notopterygium incisum (NI) from the Umbelliferae family, genuinely produced in Sichuan, China, is a classic traditional Chinese medicinal herb for treating wind-dampness arthralgia. Due to scarce natural resources, wild NI is gradually being replaced by cultivated types. However, knowledge is limited regarding the differences in chemical composition and pharmacological effects between wild and cultivated NI and between Sichuan-grown and other-region-grown NI. In this study, a plant metabolomics strategy, based on GC–MS and UHPLC-Orbitrap MS, was employed to compare metabolic profiles between wild and cultivated NI and between cultivated NI from Sichuan and cultivated NI from Gansu and Qinghai. In total, 195 metabolites were identified, and the biosynthetic pathways of coumarins and phenolic acids, which were the most abundant secondary metabolites in NI, were summarized. Additionally, seven key metabolic intermediates were uncovered, revealing the reasons for the differences in metabolic profiles between wild and cultivated NI. The anti-inflammatory effect of wild and cultivated NI was verified by inflammatory gene expression and neutrophil count using a zebrafish yolk sac inflammation model. Overall, this study presents information on the types and synthesis of pharmacodynamic substances in NI and provides a basis for its cultivation and applications.