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Hyperphosphatemia and poor uremia control are established cardiovascular risk factors in patients with end-stage renal disease (ESRD) associated with impaired endothelial dependent and independent vasodilation (EDV and EIV). Nocturnal hemodialysis [6 × 8 h/week] augments dialysis dose and offers normal phosphate (Pi) balance. We hypothesized that NHD would restore EDV (endothelial function) and EIV (vascular smooth muscle cell function) by simultaneously improving uremia and Pi control. 2 groups of ESRD patients (mean age 41 ± 2 years) stratified according to their baseline plasma Pi levels (normal Pi <1.8 mM, high Pi >1.8 mM) were studied. Dialysis dose (Kt/V per session), plasma Pi, blood pressure (BP) and brachial artery responses to reactive hyperemia (EDV), and sublingual nitroglycerin (EIV) were examined before, 1 and 2 months after conversion from conventional hemodialysis (CHD) [3 × 4 h/week] to NHD. After 2 months, NHD increased dialysis dose (from 1.24 ± 0.06 to 2.04 ± 0.08; p = 0.02) and lowered BP (from 140 ± 5/82 ± 3 to 119 ± 1/71 ± 3, p = 0.01) in all patients. In patients with adequate Pi control during CHD, EDV was normalized after 1 month of NHD. In contrast, in the high Pi group, 1 month of NHD was sufficient to reduce plasma phosphate levels, but 2 months of NHD was required for EDV to improve. Correction of uremia without normalization of plasma Pi is insufficient to improve EDV. The time course over which this improvement in vascular responsiveness occurs is delayed in hyperphosphatemic patients, suggesting that improvement in endothelial and vascular smooth muscle cell function requires restoration of normal intracellular Pi balance.