We previously reported decreased glucose-stimulated insulin secretion (GSIS) in hormone-sensitive lipase-null mice (HSL−/−), both in vivo and in vitro. The focus of the current study was to gain further insight into the signaling role and regulation of lipolysis in islet tissue. The effect of glucagon-like peptide 1 (GLP-1) on GSIS was also studied, as GLP-1 could augment GSIS via protein kinase A activation of HSL and lipolysis. Freshly isolated islets from fasted and fed male HSL−/− and wild-type (HSL+/+) mice were studied at ages 4 and 7 months. Neutral cholesteryl ester hydrolase activity was markedly reduced in islets from both 4- and 7-month-old male HSL−/− mice, whereas a marked deficiency in triglyceride lipase activity became evident only in the older mice. The deficiencies in lipase activities were associated with higher islet triglyceride content and reduced lipolysis at basal glucose levels. Lipolysis was stimulated by high glucose in islets of both wild-type and HSL-null mice. Severe deficiencies in GSIS were found, but only in islets from 7-month-old, fasted, male HSL−/− mice. GSIS was less affected in 4-month-old fasted male HSL−/− mice and not reduced in female mice. Exogenous delivery of free fatty acids (FFAs) rescued GSIS, supporting the view that the lack of endogenous FFA supply for lipid-signaling processes in HSL−/− mice was responsible for the loss of GSIS. GLP-1 also rescued GSIS in HSL−/− mice, indicating that signaling via HSL is not a major pathway for its incretin effect. Thus, the secretory phenotype of HSL-null mice is gender dependent, increases with age, and is influenced by the nutritional state. Under most circumstances, the major determinant of lipolytic flux in the β-cell involves an enzyme(s) other than HSL that is acutely activated by glucose. Our results support the view that the availability of endogenous FFA through HSL and an additional enzyme(s) is involved in providing lipid moieties for β-cell signaling for secretion in response to glucose.