Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational splicing. The structural integrity of circular sortase A and its biochemical characteristics were compared to those of the linear enzyme analog and were found to be similar under native conditions. Additionally, the modified sortase was active at concentrations of urea up to 3 M and was capable of efficient catalytic protein–protein coupling, as shown by the ligation of purified glutathione-S-transferase and green fluorescence protein. In contrast to the circular enzyme, linear sortase A was unable to mediate the ligation of substrate proteins under the same conditions. Therefore, the proposed circular sortase A has improved enzymatic properties and has applications in advanced protein engineering and design.