NADPH oxidase (NOX) is crucial for the production of reactive oxygen species (ROS) during developmental processes and stress responses across plants, animals and fungi. The interdependence of ROS and Ca2+ signals in various cellular signaling pathways is well-established, but the specific calcium channel that collaborates with NADPH oxidase to generate Ca2+-ROS co-coupling signals remains unidentified. In this study, we demonstrate that eukaryotic NOXs, such as Arabidopsis RBOHD/C/F, human NOX2 and yeast YNO1, function as calcium channels that directly mediate Ca2+ influx. Additionally, we reveal that these eukaryotic NOX family members act as non-selective cation channels, similar to human TRP family channels, capable of transporting monovalent and bivalent cations. Furthermore, RBOHD's calcium channel activity is activated by protons, and mutation of RBOHD results in significantly reduced physiological responses to low pH in a Ca2+-dependent manner. Overall, we have identified for the first time the conserved calcium channel activity of NADPH oxidases, such as plant RBOHs, and human and yeast NOXs across eukaryotic species. This discovery introduces a new paradigm for NADPH oxidase research.