The present study has investigated the transformation of sesame oil kept at low temperature during a definite period of time for refinement (called winterization) as an inactive drug ingredient by using two-dimensional difference spectra (2D-DS) analysis of spectra collected by a near-infrared (NIR) and mid-infrared (MIR) dual-wavelength spectrometer (NIR/MIR-DWS). The NIR and MIR spectra were measured nearly simultaneously from samples of sesame oil before and after winterization. The difference spectrum analysis of the obtained NIR/MIR data elucidated that, after the winterization process, the absorbances at peaks attributed to C=O, C=C, and OH groups decrease while the absorbances arising from the main chain (CH2) increase. The result indicated the removal of lignan and the fatty acids with relatively short main chains. Moreover, sesame oil unwinterized was cooled from room temperature to near 1oC and subsequently warmed to room temperature. And the cycle was repeated two times. Real-time monitoring during the cooling and warming processes were carried out using the NIR/MIR-DWS. The prediction results obtained from partial least square (PLS) calibration model for the temperature suggests that there are subtle differences in the oil composition between the first cooling process and after the warming and cooling cycle. For the more detailed analysis, two-dimensional difference spectra (2D-DS) method is proposed. The results of the analyses using 2D-DS revealed that the starting point of the transformation is around 15 oC. It can be estimated that sesame oil is mainly transformed by the first cooling down. Moreover, it was implied that the structure of methylene (CH2) was significantly related to the modifications in sesame oil with temperature change. A series of experimental results elucidated that the winterization of sesame oil removed its impurities and stabilized its conditions. These results are probably the first report on the effect of the winterization process on sesame oil.