In the course of screening for a novel cell cycle inhibitor, a potent Cdk 1 inhibitor, HY558, was found from the culture broth of Penicillium minioluteum F558 isolated from a soil sample. The molecular ion of HY558 was identified at m/z 329 (MH+) with a molecular formula of $C_20H_44ON_2$. HY558 exhibited selective antiproliferative effects on various human cancer cell lines. Its $IC_50$ values were estimated to be 0.29 mM on HepG2, 0.30 mM on HeLa, 0.30 mM on HL6O, 0.33 mM on HT-29, and 0.25 mM on AGS cells. Interestingly, Hy558 demonstrated no antiproliferative effect with normal lymphocytes used as the control, and a low level of inhibition on the proliferation of A549 cancer cells. A flow cytometric analysis of HepG2 cells revealed an appreciable arrest of cells at the G1 and G2/M phases of the cell cycle following treatment with Hy558. furthermore, DNA fragmentation due to apoptosis was observed in HeLa cells treated with 0.46 mM of HY558.