AbstractP. ginseng(Panax ginsengC. A. Meyer) is a well‐known traditional medicine that has been used for thousands of years to treat diseases. However, “ginseng abuse syndrome” (GAS) often occurs due to an inappropriate use such as high‐dose or long‐term usage of ginseng; information about what causes GAS and how GAS occurs is still lacking. In this study, the critical components that potentially caused GAS were screened through a step‐by‐step separation strategy, the pro‐inflammatory effects of different extracts on messenger RNA (mRNA) or protein expression levels were evaluated in RAW 264.7 macrophages through quantitative real‐time polymerase chain reaction (qRT‐PCR) or Western blot, respectively. It was found that high‐molecular water‐soluble substances (HWSS) significantly increased the expression of cytokines (cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL‐6)) and cyclooxygenase 2 (COX‐2) protein; gel filtration chromatography fraction 1 (GFC‐F1) further purified from HWSS showed prominent pro‐inflammatory effects by increasing the transcription of cytokines (COX‐2, iNOS, tumor necrosis factor alpha (TNF‐α), and interleukin 1β (IL‐1β)) as well as the expression of COX‐2 and iNOS protein. Moreover, GFC‐F1 activated nuclear factor‐kappa B (NF‐кB) (p65 and inhibitor of nuclear factor‐kappa B alpha (IκB‐α)) and the p38/MAPK (mitogen‐activated protein kinase) signaling pathways. On the other hand, the inhibitor of the NF‐κB pathway (pyrrolidine dithiocarbamate (PDTC)) reduced GFC‐F1‐induced nitric oxide (NO) production, while the inhibitors of the MAPK pathways did not. Taken together, GFC‐F1 is the potential composition that caused GAS through the production of inflammatory cytokines by activating the NF‐кB pathway.