| Summary: | Cancer is still the leading cause of death across the world, and there is a lack of efficient therapies. <i>Lactarius hatsudake</i> is a mushroom with a food and medicine homology that contains numerous biologically active substances. This study aimed to investigate the composition of extracts from <i>Lactarius hatsudake</i> (<i>L. hatsudake</i>) and their anti-cancer function and molecular mechanisms. Our results showed that the total phenolic content of <i>L. hatsudake</i> extracts was 139.46 ± 5.42 mg/g. The following six phenolic compounds were identified from <i>L. hatsudake</i> extracts by HPLC and UPLC-QTOF/MS: gallic acid, pyrogallol, chlorogenic acid, ferulic acid, myricetin, and cinnamic acid. Colorectal cancer cell HCT116 and hepatic cancer cell HepG2 were used to evaluate the anti-cancer function of the <i>L. hatsudake</i> extracts. Compared with HepG2 cells, the <i>L. hatsudake</i> extracts showed stronger anti-cancer activity against HCT116 cells and these were used to study molecular mechanisms. The results indicated that the <i>L. hatsudake</i> extracts could arrest the cancer cell cycle and inhibit cancer cell proliferation, which may be mediated by the MAPK/NFκB/AP-1 signalling pathway; the <i>L. hatsudake</i> extracts also promoted cancer cell apoptosis through a mitochondrial-dependent pathway. Taken together, these findings demonstrate that <i>L. hatsudake</i> ethanol extracts contain six main phenolics and illustrate the remarkable potentiality of <i>L. hatsudake</i> as a source of natural phenolics for cancer prevention and as an adjuvant in the treatment of functional foods.
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