The mushroom Agaricus blazei is studied for its nutraceutical potential and as a medicinal supplement. The aim of the present study was to investigate the chemoprotective effect of β-glucan extracted from the mushroom A. blazei against DNA damage induced by benzo[a]pyrene (B[a]P), using the comet assay (genotoxicity) and micronucleus assay with cytokinesis block (mutagenicity) in a human hepatoma cell line (HepG2). To elucidate the possible β-glucan mechanism of action, desmutagenesis or bioantimutagenesis types, three treatment protocols were tested: simultaneous, pre-treatment, and presimultaneous. The results showed that β-glucan does not exert genotoxic or mutagenic effect, but that it does protect against DNA damage caused by B[a]P in every protocol tested. The data suggest that β-glucan acts through binding to B[a]P or the capture of free radicals produced during its activation. On the other hand, the pre-treatment results also suggest the possibility that β-glucan modulates cell metabolism.