Adipose triglyceride lipase (ATGL), which catalyzes the breakdown of triglycerides in lipid droplets (LDs), plays a critical role in releasing fatty acids to support insulin secretion in pancreatic beta cells. Based on genetic downregulation of ATGL in beta cells, multiple mechanisms are proposed that acutely or chronically regulate insulin secretion. Currently, the contribution of acute versus chronic mechanisms in the regulation of insulin secretion is unclear. Also, little is known whether ATGL affects alpha cell function. Using the human-specific ATGL inhibitor, NG497, this study investigates the impact of acute inhibition of ATGL on hormone secretion from human islets. In addition, morphological differences in LDs were assessed in confocal images of beta and alpha cells. Beta cells exposed to NG497 overnight showed notable increases in LD size and number under glucose-sufficient culture. The effect of NG497 on LD accumulation in alpha cells was more prominent under fasting-simulated conditions than glucose-sufficient conditions, pointing toward a critical role for ATGL lipolysis under conditions that stimulate hormone secretion in beta and alpha cells. When exposed to NG497 acutely, human islets reduced glucose-stimulated insulin secretion mildly, particularly first-phase insulin secretion, to an extent somewhat less pronounced than the impacts of chronic ATGL downregulation. Thus, chronic mechanisms may play a predominant role in reducing insulin secretion when ATGL is downregulated. Acute exposure of human islets to NG497 significantly reduced amino acid stimulated glucagon secretion at low glucose concentration, highlighting an important potential role of ATGL lipolysis in promoting hormone secretion acutely from alpha cells.