Vascular calcification is highly prevalent in patients with chronic hemodialysis. Increased acetatemia during hemodialysis sessions using acetate-acidified bicarbonate has also been associated with several abnormalities, By contrast, these abnormalities were not induced by citrate-acidified bicarbonate dialysis. Moreover, citrate is biocompatible alternative to acetate in dialysis fluid. However, the effects of citrate on vascular calcification during hemodialysis had not been studied in detail. This study analyzed herein the effects of acetate- or citrate-acidified bicarbonate dialysis on vascular calcification. Citrate has been shown to inhibit calcification in urine in hemodialysis patients. Therefore, our hypothesis is that citrate-acidified bicarbonate dialysis could reduce vascular calcification. Blood samples before and after hemodialysis from patients on acetate- or citrate-acidified bicarbonate dialysis were collected in heparin-containing tubes (n = 35 and n = 25 respectively). To explore the effect of pre- and post-dialysis plasmatic bicarbonate and citrate on vascular calcification, rats aortic rings cultured ex vivo in Minimum Essential Medium containing 0.1% FBS and 45-calcium as radiotracer were used (n = 24). After 7 days of incubation aortic rings were dried, weighed and radioactivity was measured via liquid scintillation counting. Bicarbonate levels increase calcium accumulation in rat aortic wall in a dose-response manner (pH = 7.4). Moreover, citrate prevents calcium accumulation, with a mean inhibitor concentration (IC50) value of 733 µmol/L. During acetate-acidified bicarbonate dialysis, bicarbonate and citrate levels in plasma increase (22.29 ± 3.59 versus 28.63 ± 3.56 mmol/L; p < 0.001) and decrease (133.3 ± 53.6 versus 87.49 ± 32.3 µmol/L, p < 0.001), respectively. These changes in pos-hemodialysis plasma significantly (p < 0.001) alter calcium accumulation in the aortic wall (38.9% higher). Moreover, citrate-acidified bicarbonate dialysis increases post-hemodialysis citrate levels 5-fold (145 ± 79.8 versus 771.6 ± 184.3 µmol/L), reducing calcium accumulation in the aortic wall. Citrate-acidified bicarbonate dialysis reduces plasmatic calcium and pH variations during dialysis session (Δ[Ca2+] = -0.019 ± 0.089; ΔpH = 0.098 ± 0.043) respect to acetate-acidified bicarbonate dialysis (Δ[Ca2+] = 0.115 ± 0.118; ΔpH = 0.171 ± 0.078). To our knowledge, our study is the first to show that citrate protects against calcium accumulation in rat aortic walls ex vivo. Therefore, citrate-acidified bicarbonate dialysis may be an alternative approach to reduce calcification in hemodialysis patients without additional cost.