Targeted disruption of the lipid droplet protein, perilipin, in mice leads to constitutional lipolysis associated with marked reduction in white adipose tissue as a result of unbridled lipolysis. To investigate the metabolic adaptations in response to the constitutive lipolysis, we studied perilipin-null (plin(-/-)) mice in terms of their fatty acid oxidation and glycerol and glucose metabolism homeostasis by using dynamic biochemical testing and clamp and tracer infusion methods. plin(-/-) mice showed increased beta-oxidation in muscle, liver, and adipose tissue resulting from a coordinated regulation of the enzymes and proteins involved in beta-oxidation. The increased beta-oxidation helped remove the extra free fatty acids created by the constitutive lipolysis. An increase in the expression of the transcripts for uncoupling proteins-2 and -3 also accompanied this increase in fatty acid oxidation. Adult plin(-/-) mice had normal plasma glucose but a reduced basal hepatic glucose production (46% that of plin(+/+)). Insulin infusion during low dose hyperinsulinemic-euglycemic clamp further lowered the glucose production in plin(-/-) mice, but plin(-/-) mice also showed a 36% decrease (p < 0.007) in glucose disposal rate during the low dose insulin clamp, indicating peripheral insulin resistance. However, compared with plin(+/+) mice, 14-week-old plin(-/-) mice showed no significant difference in glucose disposal rate during the high dose hyperinsulinemic clamp, whereas 42-week-old plin(-/-) mice displayed significant insulin resistance on high dose hyperinsulinemic clamp. Despite increasing insulin resistance with age, plin(-/-) mice at different ages maintained a normal glucose response during an intraperitoneal glucose tolerance curve, being compensated by the increased beta-oxidation and reduced hepatic glucose production. These experiments uncover the metabolic adaptations associated with the constitutional lipolysis in plin(-/-) mice that allowed the mice to continue to exhibit normal glucose tolerance in the presence of peripheral insulin resistance.