This paper investigated the chemical constituents and their cytotoxicity from Penicillium jiangxiense G5A-11, a marine derived fungus isolated from a sponge in the South China Sea. Various separation methods including thin layer chromatography, silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography were used to isolate compounds from solid-rice culture of this marine derived fungus. Spectroscopic data, along with physical and chemical properties, were utilized to identify the structures of the isolated compounds, and compared with literature data. Seventeen compounds were identified from the marine fungus Penicillium jiangxiense, including N-acetyltryptamine (1), methyl 2-(6-hydroxybenzothiazol-4-yl) acetate (2), R-mevalonolactone (3), nicotinic acid (4), altechromone A (5), 2,5-dimethy- 7-hydroxychromone (6), methyl 7-hydroxy-2-methylchromone-5-carboxylate (7), stagonoculiepine [(2S,5R)-1-formyl- 1,2,3,4-tetrahydro-5H-2,5-epiminobenzo[b]azepin-5-yl acetates] (8), (4R,5S)-5-(hydroxymethyl)-5-methyl-4-(3-oxobutyl) dihydrofuran-2(3H)-one (9), 9α-hydroxy-1,2,3,4,5,10,19-heptanorergosta-7,22-diene-6,9-lact (10), 3β,5α,9α-trihydroxy- (22E,24R)-ergosta-7,22-dien-6-one (11), ergosterol peroxide(5α,8α-epidioxy-24(R)-methylcholesta-6,22-dien-3β-ol) (12), (22E,24R)-24-methylcholesta-2,22-diene-3β,5α,6β-triol (13), ergosterol (14), stigmasterol/β-sitosterol (15), 3β,5α- dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (16), and 3β,5α-dihydroxy-6β-methoxyergosta-7,22-diene (17). Among them, compounds 7 and 9 were new natural products. Cytotoxic activity test showed that compound 10 was moderately cytotoxic to human chronic myeloid leukemia cells (K562) and human gastric cancer cells (SGC-7901) with IC₅₀ values of 12.07 ± 0.12 μmol/L and 13.17 ± 0.02 μmol/L, respectively. However, the results of the anti-inflammatory activity test revealed that none of the compounds exhibited anti-inflammatory activity.