The recent accumulation of newly discovered fungal-bacterial mutualisms challenges the paradigm that fungi and bacteria are natural antagonists. To understand the mechanisms that govern the establishment and maintenance over evolutionary time of mutualisms between fungi and bacteria, we studied a symbiosis of the fungus Rhizopus microsporus (Mucoromycotina) and its Burkholderia endobacteria. We found that nonhost R. microsporus, as well as other mucoralean fungi, interact antagonistically with endobacteria derived from the host and are not invaded by them. Comparison of gene expression profiles of host and nonhost fungi during interaction with endobacteria revealed dramatic changes in expression of lipid metabolic genes in the host. Analysis of the host lipidome confirmed that symbiosis establishment was accompanied by specific changes in the fungal lipid profile. Diacylglycerol kinase (DGK) activity was important for these lipid metabolic changes, as its inhibition altered the fungal lipid profile and caused a shift in the host-bacterial interaction into an antagonism. We conclude that adjustments in host lipid metabolism during symbiosis establishment, mediated by DGKs, are required for the mutualistic outcome of the Rhizopus-Burkholderia symbiosis. In addition, the neutral and phospholipid profiles of R. microsporus provide important insights into lipid metabolism in an understudied group of oleaginous Mucoromycotina. Lastly, our study revealed that the DGKs involved in the symbiosis form a previously uncharacterized clade of DGK domain proteins.