Harvesting microalgae is a major concern for mass culture in industry. Flocculation is an easy and effective way to harvest microalgae. However, flocculation using chemical flocculants is not feasible for scaling-up due to their toxicity. As an alternative technique, mutation breeding of autoflocculating microalgae strain has been reported in this study. We characterized autoflocculating mutants of Arthrospira platensis (A. platensis) by ethyl methane sulfonate (EMS). The cells of mutants were aggregated during the culture and dry cell weight increased 1.2- to 1.8-fold compared to the wild type. Autoflocculation was induced highly at an optimal pH level of 9 and the flocculation efficiency reached almost 90%. Mutants showed higher flocculation efficiency irrespective of the addition of chemical flocculants. Thus, it is definitely useful to harvest microalgae using autoflocculating mutants in large-scale culture without any drawbacks of harvested algal biomass.