Due to efficient photosynthetic capability, robust growth, and clear genetic background, cyanobacteria are recently used for production of different biofuel and biochemical molecules by genetic engineering and showed great potentials as the next-generation microbial cell factory. For improving the production of bio-products, a number of genetic modifications are important for cyanobacteria. However, the system-level genetic modification of cyanobacteria is limited by the lack of efficient method for marker recycling. In this investigation, we introduced the self-replicable shutter vectors harboring the flipase (FLP) gene from Saccharomyces cerevisiae into two mutants of Synechocystis sp. PCC6803 and Synechococcus elongatus PCC7942 whose genomes were inserted by a kanamycin resistance gene with flipase recombination target (FRT) flanking, respectively. Transcriptional analysis by reverse transcription polymerase chain reaction showed that FLP gene was transcripted in both the two cyanobacterial strains. Genotyping analysis indicated that FLP performed its function in vivo in both two cyanobacterial strains, and the following DNA sequencing analysis on the targeted loci further confirmed that FLP exactly excised and ligated the two FRT sites between which a kanamycin resistance gene is located. The homozygous mutants free of the kanamycin resistance gene cassette were obtained by conditional expression of FLP and further dilution plating. The shuttle vectors carrying the FLP gene were then lost in these mutants by growing in the absence of antibiotics and the further single colony separation. These results demonstrate that FLP/FRT recombination system is able to be applied to the construction of markerless mutant in both Synechocystis sp. PCC6803 and S. elongatus PCC7942.