Acylation-stimulating protein (ASP) is a lipogenic hormone secreted by white adipose tissue (WAT). Male C3 knockout (KO; C3(-/-)) ASP-deficient mice have delayed postprandial triglyceride (TG) clearance and reduced WAT mass. The objective of this study was to examine the mechanism(s) by which ASP deficiency induces differences in postprandial TG clearance and body composition in male KO mice. Except for increased (3)H-labeled nonesterified fatty acid (NEFA) trapping in brown adipose tissue (BAT) of KO mice (P = 0.02), there were no intrinsic tissue differences between wild-type (WT) and KO mice in (3)H-NEFA or [(14)C]glucose oxidation, TG synthesis or lipolysis in WAT, muscle, or liver. There were no differences in WAT or skeletal muscle hydrolysis, uptake, and storage of [(3)H]triolein substrate [in situ lipoprotein lipase (LPL) activity]. ASP, however, increased in situ LPL activity in WAT (+64.8%, P = 0.02) but decreased it in muscle (-35.0%, P = 0.0002). In addition, after prelabeling WAT with [(3)H]oleate and [(14)C]glucose, ASP increased (3)H-lipid retention, [(3)H]TG synthesis, and [(3)H]TG-to-[(14)C]TG ratio, whereas it decreased (3)H-NEFA release, indicating increased NEFA trapping in WAT. Conversely, in muscle, ASP induced effects opposite to those in WAT and increased lipolysis, indicating reduced NEFA trapping within muscle by ASP (P < 0.05 for all parameters). In conclusion, novel data in this study suggest that 1) there is little intrinsic difference between KO and WT tissue in the parameters examined and 2) ASP differentially regulates in situ LPL activity and NEFA trapping in WAT and skeletal muscle, which may promote optimal insulin sensitivity in vivo.