A leucine aminopeptidase Lap1 was cloned from Aspergillus sojae GIM3.30. The truncated Lap1 without a signal peptide was over-expressed in P. pastoris, and the enzymatic characteristics of recombinant Lap1 (rLap1) were tested. The rLap1 was about 36.7 kDa with an optimal pH 8.0 and optimal temperature 50 °C for substrate Leu-p-nitroanilide and it sustained 50 % activity after 1 h incubation at 50 °C. The activity of rLap1 was significantly inhibited by EDTA, whereas Co(2+), Mn(2+), and Ca(2+) ions, but not Zn(2+) ions, restored its activity. rLap1 showed the highest activity against Arg-pNA and then Leu-, Lys-, Met-, and Phe-pNA. The 3D structure of rLap1 showed it had a conserved functional charge/dipole complex and a hydrogen bond network of Zn2-D179-S228-Q177-D229-S158 around its active center. An acidic Asp residue was found at the bottom of the substrate binding pocket, which explains its preference for basic N-terminal amino acid substrates such as Arg and Lys. rLap1 improved the degree of hydrolysis of casein and soy protein hydrolysates and also decreased their bitterness, indicating its potential utility in food production.