Proton Nuclear Magnetic Resonance (1H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshfly Sarcophaga peregrina, swallowtail butterfly Papilio xuthus, and cabbage armyworm Mamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating [13C-1]-glucose into the media, we pursued 13C-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells cultured in vitro produce ethanol from glucose as a metabolite.