We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k"c"a"t/K"M value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu^2^+ and Co^2^+ ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn^2^+ was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn^2^+, Cu^2^+ and Co^2^+ up to 90-200% of its original value, while Zn^2^+ was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.