Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are intracellular Ca 2+ -permeable cation channels whose biphasic dependence on cytoplasmic Ca 2+ gives rise to cytosolic Ca 2+ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP 3 R-mediated Ca 2+ oscillations, the structural underpinnings of the biphasic Ca 2+ dependence that underlies Ca 2+ oscillations are incompletely understood. Here, we collected images of an IP 3 R with Ca 2+ at concentrations spanning five orders of magnitude. Unbiased image analysis revealed that Ca 2+ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for free energy, we demonstrate that Ca 2+ binding at a high-affinity site allows IP 3 Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca 2+ , IP 3 Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca 2+ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca 2+ -dependence of IP 3 R channel activity.