Fat and physical inactivity are the most evident factors in the pathogenesis of obesity, and fat quality seems to play a crucial role for measures of glucose homeostasis. However, the impact of dietary fat quality on brain function, behavior, and sleep is basically unknown. In this study, mice were fed a diet supplemented with either monounsaturated fatty acids (MUFAs) or saturated fatty acids (SFAs) and their impact on glucose homeostasis, locomotion, brain activity, and sleep behavior was evaluated. MUFAs and SFAs led to a significant increase in fat mass but only feeding of SFAs was accompanied by glucose intolerance in mice. Radiotelemetry revealed a significant decrease in cortical activity in SFA-mice whereas MUFAs even improved activity. SFAs decreased wakefulness and increased non-rapid eye movement sleep. An intracerebroventricular application of insulin promoted locomotor activity in MUFA-fed mice, whereas SFA-mice were resistant. In humans, SFA-enriched diet led to a decrease in hippocampal and cortical activity determined by functional magnetic resonance imaging techniques. Together, dietary intake of MUFAs promoted insulin action in the brain with its beneficial effects for cortical activity, locomotion, and sleep, whereas a comparable intake of SFAs acted as a negative modulator of brain activity in mice and humans.