We found that fat-soluble vitamins (i.e., vitamins A, D, E and K) have novel functions such as anticancer activity, and we therefore investigated the inhibitory activity of DNA polymerases (pols) from various species by vitamins and their related compounds. In vitamin A-related compounds, fucoxanthin, astaxanthin (provitamin A), all-trans retinal (vitamin A aldehyde) and all-trans retinoic acid (vitamin A acid) inhibited the activities of mammalian replicative pols α, δ and e with IC50 values of 18-190, 14-17 and 8-30 mM, respectively, and all-trans retinol (vitamin A) did not influence any pol activities. In vitamin D-related compounds, vitamins D2 and D3 were found to be selective inhibitors of calf pol a with IC50 values of 123 and 96 μM, respectively, and provitamins D2, D3 and the active form of vitamin D3 such as 1α, 25- dihydroxyvitamin D3 could not influence any pol activities. Tocotrienols, vitamin E compounds with an unsaturated side chain with three double bonds, selectively inhibited the activity of human pol λ with IC50 values of 18-81 μM. In vitamin K compounds, vitamin K3 selectively inhibited mitochondrial pol γ activity with an IC50 value of 6 μM, although vitamins K1 and K2 did not inhibit the activities of any pols. On the other hand, no compounds tested influenced the activities of plant pols from cauliflower, prokaryotic pols, or DNA metabolic enzymes tested. These compounds suppressed the cell growth of a human gastric cancer cell line, NUGC-3, and halted at the G1 phase in the cell cycle. We discussed the molecular mechanism and relationship between pol inhibitory activity and anti-cancer activity by fat-soluble vitamins.