Force-sensing Piezo channels are essential to many aspects of vertebrate physiology. Activation of Piezo1 is facilitated by the presence of negative membrane lipids in the inner leaflet, such as phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Here, to study how Piezo1 opens, we performed molecular dynamics simulations of Piezo1 in membranes flattened by the periodic boundary effect and with or without PIP 2 lipids. The Piezo1 pore spontaneously opens in the asymmetrical bilayer but not in the symmetric membrane or when PIP 2 lipids are neutralized. Electrophysiological characterization of putative PIP 2 -interacting Piezo1 residues suggests the contribution of multiple PIP 2 binding sites. Our Piezo1 open state recapitulates ionic selectivity, unitary conductance and mutant phenotypes obtained from numerous experimental studies. Tracking ion diffusion through the open pore reveals the presence of intracellular and extracellular fenestrations, delineating a multi-fenestrated permeation pathway. This open state sheds light on the mechanisms of lipid modulation, permeation, and selectivity in a Piezo channel.