Lipid droplets (LDs) are ubiquitous organelles and the major sites of fatty acid storage and energy homeostasis in eukaryotic cells. However, despite their physiological importance, few proteins involved in the mechanism of LD formation have been identified. Here, using the model microalga Chlamydomonas reinhardtii, we show that ABHD1, a member of the α/β hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD hemi-membrane, one enzymatic and the other structural.