Abstract Background Cardiovascular disease (CVD) is responsible for 35% of total deaths worldwide. Sex hormones, such as oestrogen and progesterone, have a cardiovascular protective role in women. After menopause, women are more prone to hypertension and vascular stiffness, accelerated vascular aging and risk for CVD. Menaquinone-7 (MK-7) is a fat-soluble vitamin K2 and serves as cofactor for vitamin K-dependent protein carboxylation. The vitamin K-dependent matrix Gla protein (MGP) is synthesized in the vasculature and has shown to inhibit vascular calcification and stiffness. Purpose To investigate effects of MK-7 on vascular stiffness in pre/peri- versus post-menopausal women. Methods In a double-blind placebo-controlled clinical intervention trial (NCT02404519), 166 women (age 40-70; dephosphorylated-undercarboxylated MGP (dp-ucMGP) > 400 pmol/L at baseline) received daily 180 µg of MK-7 (n=83) or a matching placebo (n=83) for one year. Blood was sampled during intake (inclusion/exclusion), at baseline and at the end of the study to determine dp-ucMGP levels. Regional carotid-femoral (cf-PWV) and carotid-radial (cr-PWV) pulse wave velocities, vessel wall characteristics (e.g., intima-media thickness, arterial diameter, and local carotid artery PWV (caPWV), and arterial blood pressure were measured at baseline and after one year of vitamin K2 supplementation. Pre/peri- and post-menopausal women were sub-divided according to arterial stiffness (high b-stiffness index: > overall median 9.83). Results Post-hoc analyses showed a significant decrease in dp-ucMGP plasma levels of MK-7 supplemented pre/peri-menopausal (n=34; -8.58% ± 27.65; p=0.009) and post-menopausal women (n=48; -13.40% ± 27.45; p<0.001). At baseline, vascular parameters were significantly increased in post-menopausal women compared to pre/peri-menopausal women, e.g., intima media thickness (p=0.013), carotid artery diameter (p=0.007), and caPWV (p=0.012). MK-7 treatment significantly attenuated vascular stiffness in post-menopausal women (Young’s modulus; placebo +49.14% ± 77.38; MK-7 +9.38% ± 67.10; p=0.044). Post-menopausal women with a high stiffness index showed significantly improved vascular markers after MK-7 treatment, e.g., decreased blood pressure at brachialis (-3.03% ± 9.04; p=0.012), decreased blood pressure at carotid artery (-4.49% ± 11.66; p=0.008), increased distensibility coefficient (+13.25% ± 32.34; p=0.040), and increased compliance coefficient (±12.66% ± 32.64; p=0.040). Conclusions Our results confirm that hormonal changes affect the vasculature, and that post-menopausal women suffer from increased vascular stiffness reflected by functional vascular damage. Further, MK-7 supplementation significantly attenuates arterial stiffness in post-menopausal women, in which women with increased stiffness benefit most on blood pressure. Further research is needed to unravel the mechanisms by which MK-7 executes this beneficial role in post-menopausal women.