In mice lacking GPIHBP1, the lipoprotein lipase (LPL) secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why is the freshly secreted LPL in wild-type mice not captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries. We hypothesized that LPL-HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: (1) soluble GPIHBP1 in the cell culture medium; (2) GPIHBP1-coated agarose beads; and (3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained an Ly6 domain missense mutation (W109S) but was almost normal when the acidic domain of GPIHBP1 was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells.