Host cellular proteases induce influenza virus entry into cells by cleaving the viral surface envelope glycoprotein hemagglutinin (HA). However, details on the cellular proteases involved in this event are not fully available. We report here that ubiquitous type II transmembrane serine proteases, MSPL and its splice variant TMPRSS13, are novel candidates for proteases processing HA proteins of highly pathogenic avian influenza (HPAI) viruses, apart from the previously identified furin and proprotein convertases 5 and 6. HAs from all HPAI virus H5 and H7 strains have one of two cleavage site motifs, the R-X-K/R-R motif with R at position P4 and the K-K/R-K/T-R motif with K at position P4. In studies of synthetic 14-residue HPAI virus HA peptides with these cleavage site motifs, furin preferentially cleaved only HA peptides with the R-K-K-R motif in the presence of calcium and not peptides with the other motif, whereas MSPL and TMPRSS13 cleaved both types of HA peptides (those with the R/K-K-K-R motif) efficiently in the absence of calcium. Full-length recombinant HPAI virus HA with the K-K-K-R cleavage motif exhibited poor susceptibility to cleavage in the absence of MSPL or TMPRSS13 and the presence of furin in infected cells, but it was converted to mature HA subunits in transfected cells expressing MSPL or TMPRSS13, with membrane-fused giant-cell formation. This conversion and membrane fusion were suppressed by inhibitors of MSPL and TMPRSS13. Furthermore, infection with and multiplication of genetically modified live HPAI virus A/Crow/Kyoto/53/2004 (H5N1) with the K-K-K-R cleavage site motif were detected only in MSPL- and TMPRSS13-expressing cells.