Recombinant microbial transglutaminase (rMTG) is usually expressed as a soluble zymogen (pro-rMTG) in heterologous expression systems but proteolytic activation of the inactive pro-rMTG is essential. Instead of screening proteases for activating pro-rMTG, we examined an alternative method by introducing a specific cleavage site of enterokinase between the pro-peptide and mature rMTG, generating three pro-rMTG variants (Pro-mrMTG, Pro-m-rMTG and mPro-rMTG). Pro-mrMTG and Pro-m-rMTG were activated by enterokinase without degrading mature rMTG. The activation productivity of Pro-m-rMTG by enterokinase reached 92 % after 22 h activation, while the activation productivity of Pro-rMTG activated by trypsin was 47 %. MALDI-MS analysis revealed that the pro-peptide including the cleavage site was specifically removed from Pro-m-rMTG after activation. This methodology has the potential to be applied in rMTG production by incorporating highly specific cleavage sites of other proteases.