Atmospheric and room temperature plasma (ARTP) is a novel and rapid mutation tool for microbial evolution engineering, which has been proved to be convenient and powerful because of its mild operating conditions, feasible use and great impact upon the diverse alteration of microbial genomes.Spirulina platensis, a typical microalgae, is of great importance in bioindustry as a resource of carbohydrates for producing useful biofules and chemicals by fixing CO2.For the development of the microalgae bioprocess,the cell factory engineering is indispensable.In this study, we are aiming to make mutants and also tried to establish a diverse mutant library of S.platensis with altered productivity of polysaccharides, growth rate and robustness by ARTP mutation.The mutation protocol and high throughput screening method for S.platensis were firstly established.After that a mutant bank with 45% mutation rate was generated, including 25% positive mutation.The positive mutants showed certain excellent characteristics such as improved productivity of polysaccharides or growth rate when compared to the wild type.As to one outstanding mutant, the polysaccharide yield increased by 65% and showed strong flocculation which will benefit the harvest process.These mutants showed genetic stability demonstrated by more than 9 subcultures.For systematically analyzing the mutation mechanism, the typical mutants with significantly altered phenotypes were selected from the library for genomic and proteomic analysis.A plenty of mutation sites in the genome were detected by genome sequencing, and the metabolic enzymes pertaining to the metabolism of energy, glycogen, CO2 transport and photosynthesis were changed.