Cathepsin E splice variant 2 appears in a number of gastric carcinomas. Here we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variants 1 and 2 of cathepsins E, with propeptide and without it, in Escherichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant 1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by atomic force microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS) and used to rationalize its conformational properties and loss of activity.