While the plasma phylloquinone (PK) concentration is inversely correlated with cardiovascular risk, the involvement of PK in regulating endothelial function has not been directly investigated. Therefore, in this study we assessed the effects of short-term treatment with PK-deficient diets (5-10 weeks) on endothelial function in normolipidemic 14-week-old male C57BL/6JCmd mice and age-matched dyslipidaemic male E3L.CETP mice. Our results show that in normolipidemic mice dietary PK deficiency was associated with a marked reduction of PK levels in the plasma and liver (liquid chromatography-mass spectrometry measurements) and with impaired endothelium-dependent vasodilation assessed in vivo by magnetic resonance imaging (MRI). Dietary PK deficiency-induced endothelial dysfunction was fully reversed by PK supplementation. In dyslipidaemic E3L.CETP mice, dietary PK deficiency exacerbated preexisting endothelial dysfunction. Furthermore, dietary PK deficiency decreased menaquinone-4 (MK-4) levels in the aorta but did not affect blood coagulation (calibrated automated thrombography), microbiota composition (culturing and next-generation sequencing), and gut menaquinone production. In conclusion, our study demonstrated for the first time that sufficient dietary PK intake supports endothelial function in normolipidemic and dyslipidaemic mice indicating nutritional significance of dietary PK in the maintenance of endothelial function in humans.