Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A₂ (sPLA₂)-inhibitory activity. However, the mechanism by which UG inhibits sPLA₂ activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four α-helices. We previously reported that sPLA₂-inhibitory activity of UG may reside in a segment of α-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA₂ activity by binding and sequestering Ca⁺⁺, essential for sPLA₂ activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA₂-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca⁺⁺ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca⁺⁺. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA₂-inhibitory activity of UG and (ii) Ca⁺⁺-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA₂-inhibitory activity.