Matriptase is a type II transmembrane serine protease containing the non-catalytic domains (stem domain) and catalytic domain in the extra-cellular region. Our aim is to address the role of the stem domain in the interaction between matriptase and its physiological inhibitor, hepatocyte growth factor activator inhibitor type I (HAI-1). We prepared secreted variants of recombinant matriptase containing the entire extra-cellular domain (HL-matriptase) or only the catalytic domain (L-matriptase), and compared the inhibition activities of a cell membrane-anchored form of recombinant HAI-1 (maHAI-1) against the matriptase variants in the hydrolysis of peptidyl–4-methyl-coumaryl-7-amide (MCA) substrates. HL-matriptase and L-matriptase were inhibited by purified maHAI-1 with a similar extent when t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA (1) and acetyl-Lys-Thr-Lys-Gln-Leu-Arg-MCA (2) were used as substrates. However, HL-matriptase was inhibited more strongly than L-matriptase by maHAI-1 in the hydrolysis of Boc-[(2S)-2-amino-3-(benzyloxycarbonyl)propionyl]-Pro-Arg-MCA (3). These results show that the stem domain of matriptase facilitates the inhibitory interaction of this protease with maHAI-1 in the hydrolysis of substrate 3, although it has no effect in the hydrolysis of substrates 1 and 2. To our knowledge, this is the first evidence that the stem domain of matriptase can affect the interaction between this protease and HAI-1.