The steroidogenic acute regulatory protein (StAR)-related lipid transfer domain-4 (STARD4) is a sterol-binding protein that is involved in cholesterol homeostasis by intracellular sterol transport. In this work, we determined the crystal structures of human STARD4 and its Omega 1-loop mutant in apo forms at 1.95 and 1.7 angstrom resolutions, respectively. The structure of human STARD4 displays a conserved alpha-helix/beta-grip fold containing a deep hydrophobic pocket. The Omega 1-loop which serves as a lid for the hydrophobic pocket has a closed conformation. The shape of the sterol-binding cavity in the closed form is not complementary to accommodate cholesterol, suggesting that a conformational change of the Omega 1-loop is essential for sterol binding. The human STARD4 displayed sterol transfer activity between liposomes, and the mutations in the Omega 1-loop and the hydrophobic wall abolished the transfer activity. This study confirms the structural conservation of the STARD4 subfamily proteins and the flexibility of the Omega 1-loop and helix alpha 4 required for sterol transport. (C) 2019 Elsevier Inc. All rights reserved.