Objective We aimed to evaluate the immunotoxicity of type I hypersensitivity reaction of 5- hydroxymethylfurfural (5-HMF, C3H6O3 ) and its dimer 5,5′-dimethyl oxide (2-furfural) (OMBF, C12H10O5 ) in vivo and in vitro and to explore the mechanisms of action. Methods The in vivo experiments were performed on male Brown Norway (BN) rats, which were randomly divided into seven groups before a week of environmental adaptation. The groups were saline control (NS), adjuvant control (Adj.), model group (Model), low dose of 5-HMF (5-HMF-L) and high dose of 5- HMF (5-HMF-H), low dose of OMBF (OMBF-L) and high dose of OMBF (OMBF-H). BN rats in each group were given saline, adjuvant or adjuvant suspension of the corresponding concentration of drug, respectively. After 30 minutes reaction, blood was collected from the abdominal aorta, and the concentrations of IgE, IgG and IL-4 in the serum were determined by enzyme linked immunosorbent assay ( ELISA). Rat basophil leukemia ( RBL)-2H3 cells were used for the in vitro experiments. Cells were randomly divided into seven groups, as per the animal experiments. The degranulation of cells was observed by toluidine blue staining before and after administration of different concentrations of drug. The release rates of β- hexosaminidase (β-HEX) and histamine (His) in the supernatant of the cells were determined by chemiluminescence after drug administration. The mechanism of 5-HMF and its dimer OMBF-induced type I hypersensitivity was explored by Western blot analysis. Results Type I hypersensitivity can be induced by 5-HMF and its dimer OMBF at both low and high doses. The in vivo experiments demonstrated that there existed significant differences between the NS and all other groups in IgE, IgG and IL-4 in the serum, except IgG in the low dose 5-HMF group. In vitro experimental studies indicated that both the low and high dose groups showed cell degranulation, and the indicators measured showed different degrees of increase. The toluidine blue staining result showed that the degranulation of RBL-2H3 cells under the high dose of OMBF was more severe. The result of the β-HEX assay showed that the release rate of β-HEX from the supernatant of the high- dose OMBF group was higher than that of 5-HMF. The release rate of His was significantly increased when compared with the blank groups, but showed no major differences among the drug-administered groups. From the result of mitogen- activated protein kinase (MAPK) family phosphorylation levels, OMBF can promote the phosphorylation of MAPK more dramatically when compared with 5-HMF, especially in the expression of p-p38. These findings indicated that the immunotoxicity of OMBF was higher than that of 5-HMF. Conclusions Through in vitro and in vivo experimental models, we found that both 5-HMF and OMBF can induce type I hypersensitivity reactions as small molecule allergens. The levels of phosphorylated MAPKs were up-regulated, indicating that the immunotoxicity of 5-HMF and OMBF might be related to MAPK family proteins.