Chemical investigation on the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475 led to the isolation of a previously undescribed (roquefortine J, 1) and four known (2−5) roquefortine alkaloids, along with six ergosterol analogues (6−11). The planar structure of 1 was established mainly on the basis of extensive analysis of its 1D, 2D NMR, and HRESIMS spectra. The absolute configuration of 1 was determined by comparison of the calculated and experimental electronic circular dichroism spectra. Compounds 5, 6, and 7 exhibited potent anti-proliferative effects against HepG2 tumor cells with IC50 values of 7.0, 8.6, and 8.2 μM, respectively.