Entomopathogenic nematodes can be mass produced in artificial media for use as biological insecticides. Nematode in vitro media have been primarily developed on the basis of yield without fully considering nematode nutritional requirements. We investigated the quality and quantity of lipids in the entomopathogenic nematode Steinernema glaseri when grown in vivo in Popillia japonica (a natural host), Galleria mellonella (a factitious host), and in solid and liquid media. Nematode yield (infective juveniles per mg dry organic material) was 4 times higher in the in vivo compared with the in vitro cultures. Nematodes produced in vivo using P. japonica accumulated a significantly higher amount of lipids compared with nematodes grown using G. mellonella or in vitro solid and liquid methods, respectively. Fractionation of S. glaseri total lipids revealed that nematodes produced using P. japonica accumulated significantly higher phospholipids and sterols compared with other methods. C:18 fatty acids were the predominant class of lipids in S. glaseri irrespective of production method. In vivo-produced nematodes had oleic 18:1 acid as the major fatty acid, whereas in vitro-produced S. glaseri had a mixture of oleic 18:1 and linoleic 18:2 acids as the predominant fatty acids. We conclude that the lipid composition of entomopathogenic nematode is host or medium dependent. We suggest that adjusting the in vivo medium by addition of components similar to a natural host nutritional composition should improve nematode production.