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Anabaena sp. strain PCC 7120 has long served as a model organism for investigating N2-fixation, photosynthesis, and various plant-type metabolic pathways and biofuel production, as well as cellular differentiation (Xu et al., 2008, Halfmann et al., 2014, Golden and Yoon, 2003). Since more than 30,000 sequenced bacterial genomes are currently available (Land et al., 2015), specific gene inactivation and analyses of the corresponding mutant’s phenotype have become powerful tools in elucidating the function of a target gene. Here we describe a protocol to inactivate a target gene in Anabaena sp. PCC 7120 using a single-crossover approach. This approach requires only one-step cloning of an internal fragment of a target gene into an integrative vector to produce a cargo plasmid. Upon a single crossover (homologous recombination) between the cargo plasmid and the Anabaena chromosome, the endogenous target gene is disrupted by generating 3’- and 5’-deleted fragments. This gene inactivating protocol is based on an integrative vector pZR606 (Chen et al., 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.