Lipoprotein lipase (LpL) binding to heparan sulfate proteoglycans (HSPGs) is hypothesized to stabilize the enzyme, localize LpL in specific capillary beds, and route lipoprotein lipids to the underlying tissues. To test these hypotheses in vivo, we created mice expressing a human LpL minigene (hLpL^HBM) carrying a mutated heparin-binding site. Three basic amino acids in the carboxyl terminal region of LpL were mutated, yielding an active enzyme with reduced heparin binding. Mice expressing hLpL^HBM accumulated inactive human LpL (hLpL) protein in preheparin blood. hLpL^HBM rapidly lost activity during a 37°C incubation, confirming a requirement for heparin binding to stabilize LpL. Nevertheless, expression of hLpL^HBM prevented the neonatal demise of LpL knockout mice. On the LpL-deficient background hLpL^HBM expression led to defective targeting of lipids to tissues. Compared with mice expressing native hLpL in the muscle, hLpL^HBM transgenic mice had increased postprandial FFAs, decreased lipid uptake in muscle tissue, and increased lipid uptake in kidneys. Thus, heparin association is required for LpL stability and normal physiologic functions. These experiments confirm in vivo that association with HSPGs can provide a means to maintain proteins in their stable conformations and to anchor them at sites where their activity is required.