Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes which possess highly specific rRNA N-glycosidase activity and are capable of catalytically inactivating prokaryotic or eukaryotic ribosomes. Due to their unique biological activities, RIPs have been considered to have great potential in medical and agricultural applications. The cucumber genome accommodates two genes encoding type 2 ribosome-inactivating proteins, further referred to as CumsaAB1 and CumsaAB2. Type 2 RIPs, represented by ricin, usually consist of two peptides linked by a disulfide bridge. A chain with N-glycosidase activity and B chain with carbohydrate-binding activity. In this study, the expression of the cucumber RIPs was analyzed. Sequence analysis showed that CumsaAB1 is synthesized with a signal peptide and subcellular localization studies further confirmed that the protein is expressed extracellularly, following the secretory pathway. Analyses of the transcript levels in various tissues during cucumber development showed that CumsaAB1 is present at extremely low levels in most tissues while the expression of CumsaAB2 is much higher, especially in leaves from plants at first-true-leaf stage and plants at the onset of flowering. Molecular modelling of the RIP sequences was performed to unravel the three-dimensional conformation of cucumber RIPs and their carbohydrate-binding sites. This study provided valuable information on the subcellular localization, the tissue-specific expression and the structure of RIPs from cucumber plants.