As part of the safety evaluation of culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis KA21, for human consumption, we performed the bacterial reverse mutation test, the mouse micronucleus test, and mouse lymphoma test using A. brasiliensis KA21 as the test substance. The reverse mutation test utilized five bacterial strains, including Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2 uvrAO. For the micronucleus test we used mice. For the mouse lymphoma test, we used one of the most commonly used mammalian cell mutagenesis systems; the L5178YTK +/- mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base-pair changes, frameshift, and small deletions. All the tests were conducted according to the guidelines for genotoxicity testing of drugs by the Ministry of Health, Labor, and Welfare, Japan. In the bacterial reverse mutation test, no toxicity was observed up to a dose of 5,000 μg/plate. In the mouse micronucleus test, no toxicity was noted up to a dose of 1 g/kg body weight. In the mouse lymphoma test, frequency of the mutation was equal both in the presence or absence of KA21. Supporting the long history of human consumption of A. brasiliensis, the data shown in this study strongly indicate the safety of this mushroom.