The purpose of this study was to validate single-frequency hand-to-foot bioelectrical impedance analysis (HFBIA) for estimating bone mineral content (BMC) using dual-energy X-ray absorptiometry as the criterion measure in healthy men and women aged 18–40 years. A total of 80 men and women participated in this study. BMC was estimated on the same day using HFBIA and dual-energy X-ray absorptiometry. The HFBIA device provided higher mean BMC values in men and the entire sample, but not in women. A smaller standard error of estimate was observed in women (0.20, corresponding to 8% of the mean reference BMC values) compared with men (0.39, corresponding to 12% of the mean reference BMC values) and the combined sample (0.31). HFBIA provided a smaller constant error and individual estimation error indicated by the 95% limits of agreement in women (−0.05 ± 0.39) compared with men (−0.16 ± 0.78) and the entire sample (−0.10 ± 0.63). In conclusion, although BMC values were found to be more accurate in women, HFBIA overestimated BMC compared with dual-energy X-ray absorptiometry, especially in individuals with lower values. Given these results, using HFBIA to measure BMC would be inappropriate for diagnostic purposes.