Background: Trace elements (TEs) are ubiquitous and their potential interest for human health has been constantly expanding. Biological monitoring is generally considered to be a useful tool to assess human exposure to chemical agents in risk assessment both at occupational and environmental levels. However, the knowledge of accurate reference values, which may vary across countries or regions, is a prerequisite for correct interpretation of biomonitoring data. This study aimed at determining the reference distribution and the upper reference limit for 26 TEs (Al, As, Sb, Ba, Be, Bi, Cd, Cr, Co, Cu, In, Li, Mn, Hg, Mo, Ni, Pd, Pt, Pb, Se, Te, Tl, Sn, U, V, Zn) in the urine of the general adult population residing in Belgium. Methods: In total, 1022 adults not occupationally or extra-occupationally (mainly via hobbies, drugs) exposed to these TEs were recruited by occupational physicians and toxicologists according to an a priori selection procedure. Non-fasting spot urine samples were analyzed for 460 males and 541 females by inductively coupled plasma mass spectrometry (ICP-MS). Careful control was applied during collection, handling and analyses of the samples to avoid any contamination. Results: Globally, the results indicate that the exposure levels of the Belgian population to these TEs are low and grossly similar to those recently published by other national surveys. Conclusions: These new reference values and upper reference limits will be useful for future occupational and/or environmental surveys.