α- and β-glucosidases are postulated to be a very attractive therapeutic target since they catalyze the cleavage of glycosidic bonds releasing glucose from the non-reducing end of an oligo- or polysaccharide chain involved in glycoprotein biosynthesis. Glucosidase inhibitors are currently of interest owing to their promising therapeutic potential in the treatment of disorders such as diabetes, AIDS, metastatic cancer and lysosomal storage diseases. α-Glucosidases can be divided into two groups, family I and family II, and the appropriate choice of the homologue enzyme to structure-based drug design purposes could prove to be successful in the development of new pharmaceuticals. In this work, we have found the highest identity of the human enzyme with the rat intestinal sucrase, when compared to other homologue proteins commonly used in biological assays, such as Saccharomyces cerevisiae α-glucosidase. Consequently, we have built and validated a 3D model for this rat enzyme, and additionally performed docking simulations of well-known glucosidase inhibitors as well as pharmacophore modeling and molecular interaction fields studies in order to establish the most relevant structural features of inhibitors and enzyme for inhibitory activity. Using this mixed methodology we have proposed how the reported inhibitors may interact at molecular level as well as discussed the role of key α-glucosidase residues in the active site, in order to design novel proposals of inhibitors.