Glycoside hydrolases (GH) have been shown to play unique roles in various biological processes like the biosynthesis of glycans, cell wall metabolism, plant defence, signalling, and the mobilization of storage reserves. To date, GH are divided into more than 100 families based upon their overall structure. GH32 and GH68 are combined in clan GH-J, not only harbouring typical hydrolases but also non-Leloir type transferases (fructosyltransferases), involved in fructan biosynthesis. This review summarizes the recent structure-function research progress on plant GH32 enzymes, and highlights the similarities and differences compared with the microbial GH32 and GH68 enzymes. A profound analysis of ligand-bound structures and site-directed mutagenesis experiments identified key residues in substrate (or inhibitor) binding and recognition. In particular, sucrose can bind as inhibitor in Cichorium intybus 1-FEH IIa, whereas it binds as substrate in Bacillus subtilis levansucrase and Arabidopsis thaliana cell wall invertase (AtcwINV1). In plant GH32, a single residue, the equivalent of Asp239 in AtcwINV1, appears to be important for sucrose stabilization in the active site and essential in determining sucrose donor specificity.