In Escherichia coli, a long-chain acyl-CoA is a regulatory signal that modulates gene expression through its binding to a transcription factor FadR. In this study, comparative proteomic analysis of E. coli in the presence of glucose and oleic acid was performed to understand cell physiology in response to oleic acid. Among total of 52 proteins showing altered expression levels with oleic acid presence, 9 proteins including AldA, Cdd, FadA, FadB, FadL, MalE, RbsB, Udp, and YccU were newly synthesized. Among the genes that were induced by oleic acid, the promoter of the aldA gene was used for the production of a green fluorescent protein (GFP). Analysis of fluorescence intensities and confocal microscopic images revealed that soluble GFP was highly expressed under the control of the aldA promoter. These results suggest that proteomics is playing an important role not only in biological research but also in various biotechnological applications.