Triacylglycerols (TAG) serve as the predominant form of energy storage in mammalian cells, and TAG synthesis influences conditions such as obesity, fatty liver, and insulin resistance. In most tissues, the glycerol 3-phosphate pathway enzymes are responsible for TAG synthesis, and the regulation and function of these enzymes is therefore important for metabolic homeostasis. Here we review the sites and regulation of glycerol-3-phosphate acyltransferase (GPAT), acylglycerol-3-phosphate acyltransferase (AGPAT), lipin phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT) enzyme action. We highlight the critical roles that these enzymes play in human health by reviewing Mendelian disorders that result from mutation in the corresponding genes. We also summarize the valuable insights that genetically engineered mouse models have provided into the cellular and physiological roles of GPATs, AGPATs, lipins and DGATs. Finally, we comment on the status and feasibility of therapeutic approaches to metabolic disease that target enzymes of the glycerol 3-phosphate pathway. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.