Ecto-nucleotidases play a crucial role in purinergic signaling by hydrolyzing extracellular nucleotides and generating nucleosides that can be reuptaken by cells. They also produce extracellular adenosine, which acts as an agonist for P1 receptors, and inorganic pyrophosphate, which is important for bone mineralization. This review discusses four major groups of ecto-nucleotidases: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase (eN), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), and alkaline phosphatases (APs). Recent crystal structures have provided detailed insights into their spatial structures and catalytic mechanisms, enabling comparisons between these enzyme groups. The review focuses on the biochemical, cell biological, catalytic, and structural properties of these enzymes, as well as their tissue distribution and physiological and pathophysiological functions. The four groups of ecto-nucleotidases include E-NTPDases, eN, E-NPPs, and APs. E-NTPDases hydrolyze nucleoside tri- and diphosphates, while eN produces extracellular adenosine from AMP. E-NPPs hydrolyze a variety of substrates, including nucleoside triphosphates and diphosphates, and APs hydrolyze nucleoside tri-, di-, and monophosphates. The review also discusses the structure and catalytic mechanisms of these enzymes, highlighting their roles in purinergic signaling and other physiological processes. The review emphasizes the importance of these enzymes in cellular function and their potential therapeutic applications.Ecto-nucleotidases play a crucial role in purinergic signaling by hydrolyzing extracellular nucleotides and generating nucleosides that can be reuptaken by cells. They also produce extracellular adenosine, which acts as an agonist for P1 receptors, and inorganic pyrophosphate, which is important for bone mineralization. This review discusses four major groups of ecto-nucleotidases: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase (eN), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), and alkaline phosphatases (APs). Recent crystal structures have provided detailed insights into their spatial structures and catalytic mechanisms, enabling comparisons between these enzyme groups. The review focuses on the biochemical, cell biological, catalytic, and structural properties of these enzymes, as well as their tissue distribution and physiological and pathophysiological functions. The four groups of ecto-nucleotidases include E-NTPDases, eN, E-NPPs, and APs. E-NTPDases hydrolyze nucleoside tri- and diphosphates, while eN produces extracellular adenosine from AMP. E-NPPs hydrolyze a variety of substrates, including nucleoside triphosphates and diphosphates, and APs hydrolyze nucleoside tri-, di-, and monophosphates. The review also discusses the structure and catalytic mechanisms of these enzymes, highlighting their roles in purinergic signaling and other physiological processes. The review emphasizes the importance of these enzymes in cellular function and their potential therapeutic applications.