The purpose of the present study was to investigate the effects of vitamin K1 on vascular smooth muscle contractility in response to phenylephrine (Phe) during hypoxia. Rat carotid rings were placed in an organ chamber containing Krebs’ solution. The rings were subjected to hypoxia by changing the gas from 95% O2:5% CO2 to a mixture containing 95% N2:5% CO2. Concentration response curves for Phe were determined before, during, and after exposure to hypoxia. Endothelium-intact rings were incubated with vitamin K1 for 10 min in normoxic conditions before being subjected to hypoxia. In another set of experiments, endothelium-intact rings were incubated with NG-nitro-L-arginine methyl ester (L-NAME), indomethacin or a combination of these drugs for 30 min. In endothelium-intact rings, hypoxia caused significant reductions in Emax (from 0.97 ± 0.03 to 0.61 ± 0.04 g/mg; mean ± SEM) and pD2 values (from 8.26 ± 0.07 to 7.67 ± 0.10). Removal of a functional endothelium effectively prevented the hypoxia-induced reduction in Emax values, but not in pD2 values (from 9.14 ± 0.10 to 8.70 ± 0.11). Pretreatment with vitamin K1 at 3 concentrations (5 × 10–8, 5 × 10–7, 5 × 10–6 mol/l) prevented the inhibitory effect of hypoxia in intact rings. Exposure of endothelium-intact rings to L-NAME plus indomethacin also inhibited the hypoxic effect. Our results show that vitamin K1 prevents the deleterious vascular effects induced by hypoxia, probably due to its action on endothelial cells.