The present study provides iron concentrations and isotopic compositions determined by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS), along with key chemical, mineralogical and physical properties of 35 representative bulk (unfiltered) waters and bulk sediments from the Amazon River Basin. These samples from the Amazon River, five of its main tributaries (the Solimões, Negro, Madeira, Tapajós and Trombetas rivers) and four sub-tributaries (the Purus, Jaú, Ucayali and Napo rivers) were essentially collected during seven field missions conducted for over two years. These encompassed the centennial flood of May 2009 and the exceptional low water stage of September–October 2010, thereby providing the most extreme hydrological situations that have been recorded over the last hundred years. While the data confirmed massive losses of iron (up to ~ 19 000 tons/day, ca. 50% of the Amazon River bulk water budget) in the Solimões and Negro rivers mixing zone, the Fe isotope signatures of these bulk waters behaved conservatively. This property allows the use of bulk water Fe isotope signature to track iron sources and explain such isotopic signature in terms of simple mixing. Unfiltered samples from the organic-rich black water rivers present light δ⁵⁷Fe relative to the average continental crust composition. This contrasts with the composition of the bulk white waters carrying a high mineral suspended load that have δ⁵⁷Fe values undistinguishable from the crustal isotopic signature (~ 0.1‰ relative to IRMM-14). This observation indicates that the Fe isotopic composition represents a reliable direct tracer of the iron speciation and, therefore, of the host phases of iron in its sources. Specifically, the white water δ⁵⁷Fe most likely trace the signatures of igneous and sedimentary sources, as well as of their lateritic soil minerals, while the bulk black water δ⁵⁷Fe track a preferential release of Fe that has gone through a reduction step in the organic-rich horizons of tropical podzols as a result of the biological activity. This study shows that the total iron transferred by the Amazon River represents between 5 and 30% of the world's ocean Fe input by rivers, and this Amazon bulk water iron displays an isotopic composition indistinguishable from that of the average continental crust.