Nitrogen fixation in legume root nodules is very important for their agricultural performance and is beneficial to the environment. Apyrases are divalent cation-dependent, tri- and di-nucleotide phosphatases that are ubiquitously found in eukaryotes and prokaryotes. We are studying the soybean ecto-apyrase GS52, which is expressed rapidly upon rhizobial inoculation. The protein was localized to the plasma membrane. Interestingly, overexpression of GS52 in Lotus japonicus resulted in increased infection by rhizobia and higher levels of nodulation; whereas RNA interference-mediated gene silencing of GS52 markedly reduced nodulation of soybean roots. While these studies suggested an important role for GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In our recent study, the biochemical characteristics of the GS52 enzyme were investigated. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation.