Lipid transfer proteins (LTPs) are soluble plant peptides endowed with biological activities applicable in agronomy, industry, and medicine. We assessed in this work the inhibition of human and intestinal insect α-amylases by rVu-LTP. The native Vu-LTP coding sequence was inserted into pET-32 EK/LIC and produced in Escherichia coli fused with Trx and His tags (rVu-LTP-Trx-His). rVu-LTP-Trx-His was purified by Ni+-chromatography and cleaved with enterokinase to remove the tags. rVu-LTP was purified on a C18 reversed-phase column. The natural Vu-LTP was purified by chromatography steps from seeds extract. rVu-LTP translation and folding were analysed by Western blotting with antibodies against Vu-LTP and circular dichroism by comparing the spectra of Vu-LTP and rVu-LTP. Human salivary and beetles Callosobruchus maculatus and Tribolium castaneum gut α-amylases were tested for inhibition by rVu-LTP. rVu-LTP was correctly produced, translated, and folded in bacterial cytoplasm. rVu-LTP inhibited 78.1% of human salivary amylase activity at 20 μM, at 40 μM inhibited 65.7% of intestinal α-amylases activity from C. maculatus and was unable to inhibit the T. castaneum gut α-amylases. The results presented by this work suggest biotechnological application of rVu-LTP in medicine to treat obese patients and in agriculture to improve grain quality protecting plants from insect attack.