Turner syndrome (TS) is associated with increased cardiovascular risk. We investigated whether hormone replacement therapy (HRT) affects endothelial function, arterial stiffness and myocardial deformation in women with TS. Twenty-five women with TS were studied in the estrogen phase of the HRT and two months after discontinuation of HRT. The following measurements were made: flow-mediated dilation (FMD) of the brachial artery, pulse wave velocity (PWV-Complior) and central systolic blood pressure (cSBP), carotid intima-media thickness (cIMT), aortic (Ao) elastic indexes - namely Ao strain, distensibility, stiffness index and pressure strain modulus (Ep) - and left ventricular (LV) global longitudinal strain (GLS) using speckle-tracking echocardiography. Ten healthy female of similar age and BMI served as a control group. Compared to controls, women with TS on HRT had higher PWV (9.1 ± 2.4 vs. 7.5 ± 0.5 m/s), cSBP (130 ± 15 vs. 121 ± 6 mmHg), cIMT (0.66 ± 0.06 vs. 0.55 ± 0.05 mm), aortic stiffness index, Ep and LA strain, and lower FMD (7.2 ± 4 vs. 10.5 ± 2.3%), Ao strain, Ao distensibility and GLS (-18.8 ± 2.7 vs. -21.9 ± 1.5%) (P < 0.05 for all comparisons). Two months after discontinuation of HRT, all women increased FMD (11.7 ± 6 vs. 7.2 ± 4%) and reduced PWV (7.8 ± 1.7 vs. 9.1 ± 2.4 m/s) and cSBP (123 ± 14 vs. 130 ± 15 mmHg). There were no statistically significant changes in BMI, cIMT and GLS (P > 0.05 for all comparisons). The percentage decrease of cSBP was associated with the percentage decrease of PWV (r = 0.54) and reversely related with the percentage increase of FMD (r = -0.57; P < 0.05 for all comparisons). HRT in women with TS may deteriorate endothelial function contributing to increased arterial stiffness and central arterial blood pressure.