Hormone-sensitive lipase (HSL) is mainly present in adipose tissue where it hydrolyses diacylglycerol. Although expression of HSL has also been reported in the brain, its presence in different cellular compartments is uncertain, and its role in regulating brain lipid metabolism remains hitherto unexplored. We hypothesized HSL might play a role in regulating the availability of bioactive lipids necessary for neuronal function, and therefore investigated whether dampening HSL activity could lead to brain dysfunction. In mice, we found HSL protein and enzymatic activity throughout the brain, both localized within neurons and enriched in synapses. HSL-null mice were then analyzed using a battery of behavioral tests. Relative to wild-type littermates, HSL-null mice showed impaired short- and long-term memory, yet preserved exploratory behaviurs. Molecular analysis of the cortex and hippocampus showed increased expression of genes involved in glucose utilization in the hippocampus, but not cortex, of HSL-null mice compared to controls. Furthermore, lipidomics analyses indicated an impact of HSL deletion on the profile of bioactive lipids, including a decrease in endocannabinoids and eicosanoids that are known to modulate neuronal activity, cerebral blood flow, and inflammation processes. Accordingly, mild increases in the expression of pro-inflammatory cytokines in HSL mice compared to littermates were suggestive of low-grade inflammation. We conclude that HSL has a homeostatic role in maintaining pools of lipids required for normal brain function. It remains to be tested, however, whether the recruitment of HSL for the synthesis of these lipids occurs during increased neuronal activity, or whether HSL participates in neuroinflammatory responses.