The increasing use of vancomycin to treat methicillin-resistant Staphylococcus aureus (MRSA) has resulted in reduced susceptibility of MRSA to this drug. It is important to optimize vancomycin dosing in patients who are undergoing hemodialysis to attain a pre-hemodialysis serum concentration sufficient to eradicate MRSA, in accordance with recent guideline recommendations. To establish the optimal strategy for vancomycin loading dose in patients undergoing hemodialysis and to explore the determinants of pre-hemodialysis serum concentration of vancomycin measured in these patients. A prospective observational cohort study was conducted between January and June 2010. Eligible participants were adults with established stage 5 chronic kidney disease who were undergoing inpatient hemodialysis. Data were collected on loading dose administered, body weight, serum concentration of vancomycin before the subsequent hemodialysis session (pre-hemodialysis concentration), and time between end of vancomycin infusion and measurement of pre-hemodialysis serum concentration. Multivariate stepwise linear regression was performed to examine independent associations between variables and measured pre-hemodialysis serum concentration of vancomycin. Eighty-one patients were included in the study. Of 24 patients who achieved the recommended pre-hemodialysis serum concentration of vancomycin (15-20 mg/L), 14 had a loading dose between 15 and 20 mg/kg. Further analysis suggested that the pre-hemodialysis serum concentration of vancomycin was independently associated with weight-based loading dose (mg/kg) (ß = 0.293, p = 0.003), age (ß = -0.358, p < 0.001), and time between administration of the loading dose and initiation of hemodialysis (ß = -0.247, p = 0.011). The findings of this study indicate that a loading dose of 15-20 mg/kg (actual body weight) is likely to yield an optimal pre-hemodialysis serum concentration at a median elapsed time of 24 h. In addition to loading dose, patient age and time between administration of the loading dose and initiation of hemodialysis also influenced the pre-hemodialysis serum concentration of the drug.