All organisms use fatty acids (FAs) for energy substrates and as precursors for membrane and signaling lipids. The most efficient way to transport and store FAs is in the form of triglycerides (TGs); however, TGs are not capable of traversing biological membranes and therefore need to be cleaved by TG hydrolases (“lipases”) before moving in or out of cells. This biochemical process is generally called “lipolysis.” Intravascular lipolysis degrades lipoprotein-associated TGs to FAs for their subsequent uptake by parenchymal cells, whereas intracellular lipolysis generates FAs and glycerol for their release (in the case of white adipose tissue) or use by cells (in the case of other tissues). Although the importance of lipolysis has been recognized for decades, many of the key proteins involved in lipolysis have been uncovered only recently. Important new developments include the discovery of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), the molecule that moves lipoprotein lipase from the interstitial spaces to the capillary lumen, and the discovery of adipose triglyceride lipase (ATGL) and comparative gene identification-58 (CGI-58) as crucial molecules in the hydrolysis of TGs within cells. This review summarizes current views of lipolysis and highlights the relevance of this process to human disease.