Maintaining normal cellular behavior is essential for the survival of organisms. One of the main mechanisms to control cellular behavior is protein phosphorylation. The process of protein phosphorylation is reversible under the regulation of protein kinases and protein phosphatases. The importance of kinases in numerous cellular processes has been well recognized. In recent years, protein phosphatases have also been demonstrated to function actively and specifically in various cellular processes and thus have gained more and more attention from researchers. In the animal kingdom, regeneration frequently occurs to replace or repair damaged or missing tissues. Emerging evidence has revealed that protein phosphatases are crucial for organ regeneration. In this review, after providing a brief overview of the classification of protein phosphatases and their functions in several representative developmental processes, we highlight the critical roles that protein phosphatases play in organ regeneration by summarizing the most recent research on the function and underlying mechanism of protein phosphatase in the regeneration of the liver, bone, neuron, and heart in vertebrates.