The green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum (previously known as Chlorobium tepidum), which grows at an optimal temperature of around 45 °C, biosynthesized unique disaccharide rhamnosylgalactosyldiacylglyceride (RGDG) having a methylene-bridged palmitoleyl (17:Cyc) and a palmitoyl group (16:0) as the two acyl chains in a molecule [RGDG(17:Cyc,16:0)], together with the corresponding monosaccharide monogalactosyldiacylglyceride (MGDG). Here, we report changes in the structure and composition of the glycolipids that are dependent upon the temperature and period of cultivation. With a decrease in temperature to 25 °C, the two major glycolipids were almost completely eliminated, and MGDG with a palmitoleyl (16:1) and a (16:0) group concomitantly became the major glycolipid. MGDG(16:1,16:0) corresponded to the removal of an α-rhamnosyl and a cyclopropyl methylene group from RGDG(17:Cyc,16:0) and the lack of the CH(2) group in MGDG(17:Cyc,16:0). The structural conversion was almost reversible when the Cba. tepidum adapted to low and high temperatures was cultured again at 45 and 25 °C, respectively. Moreover, during this cultivation, the structure and composition of glycolipids were sequentially changed: MGDG(16:1,16:0), MGDG(17:Cyc,16:0), and RGDG(17:Cyc,16:0) predominated in the exponential, stationary and late phases of the cultivation, respectively. On the basis of these time-dependent changes, the unique disaccharide RGDG(17:Cyc,16:0) was thought to be created by the site-specific transfer of an α-rhamnosyl group to MGDG(17:Cyc,16:0) after insertion of a methylene group into the precursor MGDG(16:1,16:0). These culturing temperature- and time-dependent changes in glycolipids at the molecular level allow us to discuss their biosynthesis as well as physiological function in green photosynthetic bacteria.