Plant invertases are sucrolytic enzymes essential for plant metabolism and development. Enzyme activity is regulated on a posttranslational level via inhibitory proteins, referred to as invertase inhibitors. Ectopic expression of invertase inhibitors in crop plants has high biotechnological potential. However, little biochemical and up to now no detailed structural information is available about this class of plant regulatory proteins. Here, we present the crystal structure of the cell wall-associated invertase inhibitor Nt-CIF from tobacco at a resolution of 1.87A. The structural model reveals an asymmetric four-helix bundle with an uncommon N-terminal extension that appears to be critical for the structural integrity of the protein. Structure analysis of a second crystal form grown in the presence of CdCl(2) reveals two metal binding sites. Nt-CIF is highly thermostable and retains full inhibitory activity after cooling to ambient temperatures. The structure of Nt-CIF provides the first three-dimensional information source for the posttranslational regulation of plant invertases. Based on the recently discovered sequence homology between inhibitors of invertases and pectin methylesterases, our structural model is likely to represent a scaffold also used for the regulation of the latter enzymes, which do not share sequence similarity with invertases. Thus, our structural model sets the 3D-stage for the investigation of posttranslational regulation of invertases as well as pectin methylesterases.