We investigated the chemical constituents and anti-tumor activity of cultivated Pholiota adiposa in vitro using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. HepG-2, A549, HeLa, and MCF-7 cells, which are 4 kinds of human cancer cell lines, were cultured in vitro, treated with different concentrations of the ethanol extract of Ph. adiposa (EPA), and cytotoxicity was determined using the cell counting kit-8 assay. Flow cytometry was used to analyze the apoptosis of HepG-2 cells via annexin V-fluorescein isothiocyanate/propidium iodide double staining. Expression levels of apoptosis-associated proteins were determined via Western blotting analysis. Thirty-five components were consistent with those recorded in the chemical composition database, with sterols, fatty acids, and polysaccharide compounds accounting for a relatively high proportion. EPA showed the strongest cytotoxicity against HepG-2 cells, increasing the apoptosis rate up to 23.71 ± 1.59% at a concentration of 50 μg/mL. Ph. adiposa has various functional chemical constituents and potential anti-tumor applications. We found that the functional constituents exerted anti-tumor activity by inducing apoptosis. Furthermore, the expression levels of BCL-2-associated X were increased, whereas those of BCL-2 were decreased in cells after treatment with EPA. These results suggest that EPA induces HepG-2 cell apoptosis via a caspase-mediated pathway.