α-amylases are ubiquitous enzymes belonging to the glycosyl hydrolase (GH13) family, whose members share a high degree of sequence identity, even between distant organisms. To understand the determinants of catalytic activity of α-amylases throughout evolution, and to investigate the use of homologous enzymes as a model for the human one, we compared human salivary α-amylase, Aspergillus oryzae α-amylase and pancreatic porcine α-amylase, using a combination of in vitro and in silico approaches. Enzyme sequences were aligned, and structures superposed, whereas kinetics were spectroscopically studied by using commercial synthetic substrates. These three enzymes show strikingly different activities, specifically mediated by different ions, despite relevant structural homology. Our study confirms that the function of α-amylases throughout evolution has considerably diverged, although key structural determinants, such as the catalytic triad and the calcium-binding pocket, have been retained. These functional differences need to be carefully considered when α-amylases, from different organisms, are used as a model for the human enzymes. In this frame, particular focus is needed for the setup of proper experimental conditions.