The first amphibian skin secretion-derived Bowman-Birk type chymotrypsin inhibitor is described here from the Asian green frog, Hylarana erythraea, and was identified by use of molecular cloning and tandem mass spectrometric amino acid sequencing. It was named Hylarana erythraea chymotrypsin inhibitor (HECI) and in addition to inhibition of chymotrypsin (Ki = 3.92 ± 0.35 μM), the peptide also inhibited the 20 S proteasome (Ki = 8.55 ± 1.84 μM). Additionally, an analogue of HECI, named K9-HECI, in which Phe9 was substituted by Lys9 at the P1 position, was functional as a trypsin inhibitor. Both peptides exhibited anti-proliferation activity against the human cancer cell lines, H157, PC-3 and MCF-7, up to a concentration of 1 mM and possessed a low degree of cytotoxicity on normal cells, HMEC-1. However, HECI exhibited higher anti-proliferative potency against H157. The results indicate that HECI, inhibiting chymotryptic-like activity of proteasome, could provide new insights in treatment of lung cancer.