In this study, an antimicrobial peptide composed of three tandem repeats of Mytichitin-A (3 x Mytichitin-A) with a designed molecular weight of approximately 25 kDa was expressed in the green alga Chlamydomonas reinhardtii. The yield of 3 x Mytichitin-A reached 0.28% of the total soluble protein of the 3 x Mytichitin-A-expressing transgenic cells and the expression level was stable following continuous passaging of the cells for six months. Compared to its natural and yeast-produced recombinant counterparts, which showed a very low level of growth inhibition of gram-negative bacteria, the 3 x Mytichitin-A inhibited the growth of gram-negative bacteria at a minimum inhibition concentration value ranging between 60 and 80 mu g/ml. The expressed 3 x Mytichitin-A did not show toxicity to HEK293 cells. Its bioactivity was hardly affected by temperature and pH but was impaired to some extent by the proteinase treatment. Taken together, our study showed that C reinhardtii can be used as a cellular factory to produce bioactive Mytichitin-A in a multimeric format. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.