Acylation modifications play a central role in biological and physiological processes. Across a range of biomolecules from phospholipids to triglycerides to proteins, introduction of a hydrophobic acyl chain can dramatically alter the biological function and cellular localization of these substrates. Amongst the enzymes catalyzing these modifications, the membrane boundO-acyltransferase (MBOAT) family occupies an intriguing position as the combined substrate selectivities of the various family members span all three classes of these biomolecules. MBOAT-dependent substrates are linked to a wide range of health conditions including metabolic disease, cancer, and neurodegenerative disease. Like many integral membrane proteins, these enzymes have presented challenges to investigation due to their intractability to solubilization and purification. However, over the last several years new solubilization approaches coupled with computational modeling, crystallography, and cryoelectron microscopy have brought an explosion of structural information for multiple MBOAT family members. These studies enable comparison of MBOAT structure and function across members catalyzing modifications of all three substrate classes, revealing both conserved features amongst all MBOATs and distinct architectural features that correlate with different acylation substrates ranging from lipids to proteins. We discuss the methods that led to this renaissance of MBOAT structural investigations, our new understanding of MBOAT structure and implications for catalytic function, and the potential impact of these studies for development of new therapeutics targeting MBOAT-dependent physiological processes.