Phellinus linteus, also known as the sanghuang mushroom, is a medicinal mushroom that has been recognized as beneficial to health for more thousands of years.Among its diverse valuable secondary metabolites, the yellow-brown styrylpyrone pigment hispidin has garnered significant attention due to its various pharmacological effects. However, recently after detailed morphological and molecular phylogenetic studies, the correct scientific name of the true sanghuang strains was shown not to be P. linteus but Sanghuangporus sanghuang. As the incorrect binomial name P. linteus has long been misleadingly referred, there is a need to evaluate the safety of S. sanghuang. Moreover, the growing conditions can impact the secondary metabolite profile of the fungi. Hence, this study is the first to optimize hispidin production and to investigate the genotoxic and oral toxic effects of hispidin-enriched S. sanghuang mycelia. In order to induce the biosynthesis of hispidin, 15 different culture media consisting of five carbon sources, five nitrogen sources, and five initial pH conditions were screened. Glucose and yeast extract at an initial pH of 5 were found to be the most suitable carbon and nitrogen sources, respectively, for the optimal growth and production of hispidin. Moreover, the production of hispidin was 3 mg/g in a 20-ton bioreactor under optimal conditions. Furthermore, the ames test, in vitro chromosome aberration test, acute oral toxicity test, and bone marrow micronucleus test were used to detect toxicological properties of 3 mg/g hispidin-enriched S. sanghuang mycelia. In all tests, there was no statistically significant difference between the mycelia and the negative control. Based on the results obtained, the present study demonstrates that 3 mg/g hispidin-enriched S. sanghuang mycelia has a very low order of toxicity, which supports its safety for human consumption. K E Y W O R D S hispidin, Phellinus linteus, safety assessment, Sanghuangporus sanghuang, toxicity | 1865 LI et aL.