These files contain data supporting all results in Ehrlich et. al. "The gating loop controls Pannexin 1 channel opening in living cells." In Ehrlich et. al. we found: Pannexin1 (Panx1) is an ATP release channel with diverse roles in immune signaling and neurotransmission. While the irreversible activation of Panx1 during cell death is well characterized, how the channel reversibly opens in living cells has remained unclear. Here, we present cryo‐EM structures of Panx1 in the presence of lysophospholipids, a recently identified reversible endogenous activator. These structures reveal an open conformation distinct from that induced by caspase‐mediated cleavage, in which both the N‐terminal helix and a previously unrecognized structural element, termed the gating loop, undergo coordinated rearrangements. Site‐directed mutagenesis and electrostatic free‐energy calculations indicate that these conformational changes create a pore environment favorable for ion permeation. Notably, gating‐loop movement alone is sufficient to promote channel opening, defining a reversible activation mechanism for Panx1 in living cells.