Protoporphyrinogen oxidase having an activity of imparting acifluorfen resistance and gene thereof are provided. Cyanobacterium protoporphyrinogen oxidase gene is identified by introducing a protoporphyrinogen oxidase gene of Arabidopsis into cyanobacterium, disrupting a cyanobacterium gene with a transposon, selecting a mutant strain in which protoporphyrinogen oxidase gene is disrupted, identifying the disrupted protoporphyrinogen oxidase gene, and isolating the disrupted protoporphyrinogen oxidase gene. This procedure is effective as a gene isolation technique when a protein derived from other organism species that is homologous to a known protein (e.g., protoporphyrinogen oxidase from cyanobacterium) can not be found in a gene database of the other species.