The Lost City Hydrothermal Field hosts a distinctive microbial ecosystem that is supported by the products of serpentinization reactions. The calcium carbonate chimneys here contain abundant isoprenoidal and non-isoprenoidal ether lipids, the structural diversity of which is similar to that found in carbonate crusts at cold seeps where anaerobic oxidation of methane (AOM) is the dominant biogeochemical process. The microbial community at Lost City includes abundant archaea, which largely belong to a single phylotype within the methanogenic Methanosarcinales. Isoprenoidal diethers derived from these archaea have polar head groups comprising phosphatidylglycerol or monoglycosyl moieties, although many isoprenoidal diethers detected in these carbonates lack head groups. The non-isoprenoidal diether lipids at Lost City are largely glycolipids. Glycosydic non-isoprenoidal diethers have not previously been reported from any environments or bacterial isolates. Glycolipids are common in archaea where they contain isoprenoidal hydrocarbon cores ether linked to glycerol. Glycolipids also occur in some bacteria and in these non-isoprenoidal fatty acid chains are ester linked to glycerol. However, the glycosylated non-isoprenoidal diether lipids at Lost City contain a previously undetected combination of archaeal and bacterial traits that might be an adaptive response to the vent environment. We hypothesize that utilization of glycosyl head groups instead of phosphatidyl head groups is a strategy for conservation of phosphate by organisms growing in fluids low in this essential nutrient.