In hemodialysis patients, lower plasma magnesium (Mg) concentrations are associated with a higher overall and cardiovascular mortality. The optimal concentration appears to be above the reference range for the healthy population. Plasma Mg is not routinely measured after hemodialysis. Aim of this study was to determine the effect of routine hemodialysis on plasma Mg. Plasma Mg was measured in duplicate before (Mgpre) and after (Mgpost) dialysis in 6 consecutive hemodialysis sessions of 34 patients using a fixed 0.50 mmol/L dialysate Mg concentration. Mean Mgpre was 0.88 mmol/L (±0.14) and mean Mgpost was statistically significantly lower: mean intra-dialytic decline 0.10 mmol/L (95%-CI 0.06-0.13). A 0.10 mmol/L higher Mgpre was associated with a 0.03 mmol/L higher Mgpost (95%-CI 0.024-0.037). At a Mgpre of 0.74 mmol/L, Mgpost equalled Mgpre. There was an intra-dialytic decline of plasma Mg at higher Mgpre values and an increase at lower Mgpre values. In conclusion, in the majority of the hemodialysis patients, Mgpre concentrations are in the reference range of the healthy population, which may be too low for hemodialysis patients. Routine hemodialysis with the widely used 0.50 mmol/L dialysate Mg concentration, further declines magnesium in the majority of patients. Current dialysate Mg concentrations may be too low.