Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new alpha-amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of alpha-amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic alpha-amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.