Although human pannexins (PanX) are homologous to gap junction molecules, their physiological function in vertebrates remains poorly understood. Our results demonstrate that overexpression of PanX1 results in the formation of Ca²⁺-permeable gap junction channels between adjacent cells, thus, allowing direct intercellular Ca²⁺ diffusion and facilitating intercellular Ca²⁺ wave propagation. More intriguingly, our results strongly suggest that PanX1 may also form Ca²⁺-permeable channels in the endoplasmic reticulum (ER). These channels contribute to the ER Ca²⁺ leak and thereby affect the ER Ca²⁺ load. Because leakage remains the most enigmatic of those processes involved in intracellular calcium homeostasis, and the molecular nature of the leak channels is as yet unknown, the results of this work provide new insight into calcium signaling mechanisms. These results imply that for vertebrates, a new protein family, referred to as pannexins, may not simply duplicate the connexin function but may also provide additional pathways for intra- and intercellular calcium signaling and homeostasis.