Studies conducted in China linked selenium deficiency to higher risk of esophageal squamous cell carcinoma (ESCC), but this has not been widely tested outside that selenium‐deficient region. The aim of this study was to investigate the association between selenium and other mineral concentrations in toenails and risk of ESCC in a region with high incidence rates. In this nested case–control study, we identified 222 cases of ESCC from the Golestan Cohort Study, Iran, which has followed up 50,045 participants since enrollment (2004–2008). We randomly selected one control for each case matched by age and sex, using incidence density sampling. We used toenail samples collected at baseline to measure the concentration of selenium, zinc, chromium, mercury, and scandium using instrumental neutron activation analysis. Multivariate adjusted logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals. Median nail selenium, zinc, chromium, and mercury levels were 1.01, 74.59, 0.77, and 0.018 μg/g in cases and 1.02, 75.71, 0.71, and 0.023 μg/g in controls, respectively. The adjusted odds ratios comparing each fourth quartile of mineral status versus the first quartile were as follows: selenium = 0.78 (95% CI, 0.41–1.49); zinc=0.80 (95% CI, 0.42–1.53); chromium = 0.91 (95% CI, 0.46–1.80); and mercury=0.61 (95% CI, 0.27–1.38), and all trend tests were non–significant. The nail selenium concentration in our controls reflects relatively high selenium status. No evidence of association between selenium or chromium concentrations in toenails and the risk of ESCC was detected in this population.