We have screened the inhibitors of mammalian DNA polymerases from natural products, and in the process found that either sulfoglycolipids or sulfoquinovosyl monoacylglycerol with a C18-saturated fatty acid (C18-SQMG), potently and selectively inhibited the activity of mammalian DNA polymerase (pol) and moderately the pol alpha. C18-SQMG was a cancer cell growth suppressor and a promissive anti-tumor agent. The purpose of this study was to elucidate the cell growth inhibition mechanism of C18-SQMG using HeLa cells. Analyses of the cell cycle and cyclin expression suggested that C18-SQMG arrested the cell cycle at intra-S phase, and the inhibition manner of DNA replication by C18-SQMG was similar to that by hydroxyurea. However, the DNA replication block by C18-SQMG did not induce degradation of Cdc25A protein, which was required for the replication block by hydroxyurea. C18-SQMG somewhat delayed mitosis because it induced phosphorylation of protein kinases, such as checkpoint kinases 1 and 2. These results suggest that C18-SQMG at first blocked DNA replication at the S phase by inhibiting replicative DNA polymerases, such as alpha, and then as the result of the inhibition, the other checkpoint signals associated with the pol might have responded.