The ghrelin-producing ε cell represents the fifth endocrine cell type in human pancreatic islets. The abundance of ε cells in adult pancreas is extremely low, which has hampered the investigation on the molecular pathways regulating the development and the function of this cell type. In this study, we explored the molecular features defining the function of pancreatic ε cells isolated from adult nondiabetic donors using single-cell RNA sequencing technology. We focus on transcription factors, cell surface receptors, and genes involved in metabolic pathways that contribute to regulation of cellular function. Furthermore, the genes that separate ε cells from the other islet endocrine cell types are presented. This study expands prior knowledge about the genes important for ε cell functioning during development and provides a resource to interrogate the transcriptome of this rare human islet cell type.