Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline pKa (pKe1 and pKe2) values of about 4 and 10. The ionizable group for pKe2 was assigned to Lys or Arg by thermodynamic analysis; however, no such residues are present in the active site. Hence, based on the crystal structure, we hypothesized that a water molecule bound to the main-chain nitrogen of Ala162 (W1) or the main-chain carbonyl oxygen of Pro217 (W2) is a candidate for the ionizable group for pKe2 [Takeharu, H. et al. (2011) Biochim. Biophys. Acta 1814, 1940–1946]. In this study, we inspected this hypothesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu–Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2, all 19 variants, in which one of all Lys and Arg residues was replaced by Ala, retained activity, indicating that neither Lys nor Arg is the ionizable group. pKe2 values of A162S, A162V and A162G were 9.6 ± 0.1, 9.5 ± 0.1 and 10.4 ± 0.2, respectively, different from that of wild-type MMP-7 (WT) (9.9 ± 0.1) by 0.3–0.5 pH unit, and those of P217S, P217V and P217G were 10.1 ± 0.1, 9.8 ± 0.1 and 9.7 ± 0.1, respectively, different from that of WT by 0.1–0.2 pH unit. These results suggest a possibility of W1 or W2 as the ionizable group for pKe2.