Serum selenium levels have been thought to be decreased in hemodialysis patients; however, results of previous studies have been inconsistent. Population-based hemodialysis patients (n = 1,041) and randomly recruited healthy controls (n = 384) were enrolled. Serum selenium levels were determined by inductively coupled plasma mass spectrometry and compared in hemodialysis patients and controls using analysis of covariance after adjustment for confounding factors with p < 0.1 as the result of the multiple regression analysis. Age, serum albumin levels, hsCRP levels, LDLC levels, HDLC levels, regular drinking habit and hemodialysis treatment were significantly associated with serum selenium levels in multiple regression analysis. Multivariate-adjusted means (95% CIs) of serum selenium levels were 103 µg/l (101–105) in hemodialysis patients and 117 µg/l (114–121) in controls. Selenium levels in hemodialysis patients were decreased. Whether decreased serum selenium levels contribute to increased risks for morbidity and mortality in hemodialysis patients should be examined.