Exposure to chemicals in workplace settings leads to significant occupational diseases related to hypersensitivity reactions. In recent years, diacetyl which is used as a food additive is thought to be as an important hazard due to its sensitization potency for worker's health. Therefore, in this study, we aimed to investigate the sensitization potency of diacetyl for the purpose of its hazard evaluation. Nonradioactive ex vivo local lymph node assay: BrdU-enzyme-linked immunosorbent assay (ELISA) method with short-term and long-term exposure protocols were conducted based on animal welfare principles. As end points, lymphocyte proliferation, cytokine releases, and total serum IgE levels were measured by ELISA method. After short-term dermal exposure to diacetyl, primary Th1 cytokine interleukin-2 (IL-2) and Th2 cytokines IL-4 and IL-13 levels were significantly increased relatively to vehicle control, whereas such significant increases were not observed in long-term exposure. According to our measurements of IgE levels after long-term dermal exposures to chemicals, diacetyl led to significant increase. In conclusion, the findings that showed significant increases in IL-13 and total serum IgE levels induced with diacetyl can be relevant to respiratory sensitizing hazard of this chemical.