Background: Anaphylactoid reactions induced by preparations containing red ginseng have been reported. The aim of this study is to evaluate the allergenicity and screen potential allergens in red ginseng extract thoroughly. Methods: Red ginseng extract (RGE) and diferent fractions of RGE were prepared and evaluated by measuring the degranulation and viability of rat basophilic leukemia 2H3 (RBL-2H3) cells. Potential allergens were screened by RBL-2H3 cell extraction and allergenicity verifed in RBL-2H3 cells, mouse peritoneal mast cells, Laboratory of Allergic Disease 2 (LAD2) human mast cells and mice, respectively. Results: 80% ethanol extract of red ginseng extract induced mast cell degranulation with less cytotoxicity, but 40% ethanol extract could not. Ginsenoside Rd and 20(S)-Rg3 could induce a signifcant increase in β-hexosaminidase release, histamine release and translocation of phosphatidylserine in RBL-2H3 cells. Ginsenoside Rd and 20(S)-Rg3 also increased β-hexosaminidase release and the intracellular Ca2+ concentration in mouse peritoneal mast cells and LAD2 cells. In addition, histamine levels in serum of mice were elevated dose-dependently. Conclusions: Ginsenoside Rd and 20(S)-Rg3 are potential allergens that induce the release of mediators associated with anaphylactoid reactions. Our study could guide optimization of methods associated with Rd/20(S)-Rg3-containing preparations and establishment of quality standards for safe application of Traditional Chinese Medicines.