Cell-free protein synthesis utilizes the translational machinery in a cell extract. Unlike the conventional cell-based expression methods, not being affected by the conditions for cell growth, cell-free protein synthesis enables flexible manipulation of individual factors affecting the efficiency protein biosynthesis. However, the high cost and low stability of the energy sources to regenerate ATP have limited the use of cell-free synthesis for large-scale production of recombinant proteins. One of the approaches to address this problem is to use glucose as an alternative energy source to regenerate ATP through the glucose-metabolizing pathways in a cell extract. In this study, in an attempt to improve the efficiency of ATP regeneration by reinforcing oxidative phosphorylation process, we supplemented with cellular lipids to a glucose-fueled reaction mixture for cell-free protein synthesis. As a result of the lipid supplementation, the productivity of chloramphenicol acetyltransferase in a cell-free synthesis system using glucose increased more than 6 fold compared to when the lipid was not supplemented.