Soluble inorganic pyrophosphatases (PPases) comprise two evolutionarily unrelated families (I and II). These two families have different specificities for metal cofactors, which is thought to be due to the fact that family II PPases have three active site histidines whereas family I PPases have none. Here, we report the structural and functional characterization of a unique family I PPase from Mycobacterium tuberculosis (mtPPase) that has two His residues (His21 and His86) in the active site. The 1.3-Å three-dimensional structure of mtPPase shows that His86 directly interacts with bound sulfate, which mimics the product phosphate. Otherwise, mtPPase is structurally very similar to the well-studied family I hexameric PPase from Escherichia coli, although mtPPase lacks the intersubunit metal binding site found in E. coli PPase. The cofactor specificity of mtPPase resembles that of E. coli PPase in that it has high activity in the presence of Mg²⁺, but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn²⁺ or Zn²⁺. Replacements of His21 and His86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg²⁺- and Mn²⁺-supported reactions (H86K) or reduced Mg²⁺-supported activity (H21K). However, both replacements markedly increased the Zn²⁺-supported activity of mtPPase (up to 11-fold). In the double-mutant, Zn²⁺ was a 2.5-fold better cofactor than Mg²⁺. These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity.