Three-disulfide-bridged Ecballium elaterium trypsin inhibitor II (EETI-II) is a 28-residue peptide that belongs to the squash family of canonical trypsin inhibitors. Herein, we report synthesis and biological activity of three EETI-II analogs. In each of analog, a pair of cysteine residues forming a native disulfide bridge was individually replaced by a pair of selenocysteine residues. All selenopeptide analogs were chemically synthesized using the Fmoc protocol and subsequently folded in the presence of oxidized and reduced glutathione. The analogs containing a diselenide bridge displayed association constants with trypsin that ranged from 2.6 × 109 to 5.1 × 109 [M−1]. Our results suggest that the selenopeptide analogs retained low nanomolar inhibitory potencies, and only the diselenide bridge adjacent to the inhibitory binding loop weakened the interactions with trypsin by approximately fivefold. We discuss these findings in the context of a broader use of selenopeptide analogs as proxies to study cysteine-rich peptides.