Ecto-nucleotidases play a crucial role in purinergic signal transmission by hydrolyzing extracellular nucleotides, controlling their availability at P2 receptors and generating extracellular nucleosides for cellular reuptake. They also produce and hydrolyze extracellular 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, allowing predictions of their catalytic processes and comparisons between enzyme groups. The review focuses on the biochemical, cell biological, catalytic, and structural properties of these enzymes, including tissue distribution and physiological and pathophysiological functions. Ecto-nucleotidases are involved in purinergic signaling, bone mineralization, and other processes, with varying substrate specificities and catalytic properties. The review also discusses the role of protein interactions and the impact of glycosylation on enzyme activity.Ecto-nucleotidases play a crucial role in purinergic signal transmission by hydrolyzing extracellular nucleotides, controlling their availability at P2 receptors and generating extracellular nucleosides for cellular reuptake. They also produce and hydrolyze extracellular 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, allowing predictions of their catalytic processes and comparisons between enzyme groups. The review focuses on the biochemical, cell biological, catalytic, and structural properties of these enzymes, including tissue distribution and physiological and pathophysiological functions. Ecto-nucleotidases are involved in purinergic signaling, bone mineralization, and other processes, with varying substrate specificities and catalytic properties. The review also discusses the role of protein interactions and the impact of glycosylation on enzyme activity.