For the first time in animal evolution, the emergence of gap junctions allowed direct exchanges of cellular substances for communication between two cells. Innexin proteins constituted primordial gap junctions until the connexin protein emerged in deuterostomes and took over the gap junction function. After hundreds of millions of years of gene duplication, the connexin gene family now comprises 21 members in the human genome. Notably, GJA1, which encodes the Connexin43 protein, is one of the most widely expressed and commonly studied connexin genes. The loss of Gja1 in mice leads to swelling and a blockage of the right ventricular outflow tract and death of the embryos at birth, suggesting a vital role of Connexin43 gap junction in heart development. Since then, the importance of Connexin43-mediated gap junction function has been constantly expanded to other types of cells. Other than forming gap junctions, Connexin43 can also form hemichannels to release or uptake small molecules from the environment or even mediate many physiological processes in a gap junction-independent manner on plasma membranes. Surprisingly, Connexin43 also localizes to mitochondria in the cell, playing important roles in mitochondrial potassium import and respiration. At the molecular level, Connexin43 mRNA and protein are processed with very distinct mechanisms to yield carboxyl-terminal fragments with different sizes, which have their unique subcellular localization and distinct biological activities. Due to many exciting advancements in Connexin43 research, this review aims to start with a brief introduction of Connexin43 and then focuses on updating our knowledge of its gap junction-independent functions.