The biological cycle of rock-derived nutrients on the continents is a major component of element transfer between the Earth's surface compartments, but its magnitude currently remains elusive. The use of the stable isotope composition of rock-derived nutrients, which can be fractionated during biological uptake, provides a promising path forward with respect to quantifying biological cycling and its overall contribution to global element cycling. In this paper, we rely on the nutrient-like behaviour of the trace element barium (Ba) and use its elemental and stable isotope compositions in dissolved and sediment load river samples to investigate biological cycling in the Amazon Basin. From these measurements, we show that dissolved Ba mainly derives from silicate rocks, and a correlation between dissolved Ba and K abundances suggests that biological cycling plays a role in the Ba river budget. Furthermore, the isotope composition of Ba (δ138Ba) in the dissolved load was found to be significantly different from that of the parent silicate rocks, implying that dissolved Ba isotopic signatures are affected by (i) the precipitation of soil-forming secondary phases as well as (ii) biological uptake and release from dead organic matter. Results from an isotope mass balance method applied to the river dissolved load data indicate that, after its release to solution by rock weathering, Ba is partitioned between the river dissolved load, secondary weathering products (such as those found in soils and river sediments), and the biota. In most sub-catchments of the Amazon, river Ba abundances and isotope compositions are significantly affected by biological cycling. Relationships between estimates of Ba cycled through biota and independent metrics of ecosystem dynamics (such as gross primary production and terrestrial ecosystem respiration) allow us to discuss the role of environmental parameters such as climate or erosion rates on the biological cycling of Ba and, by extension, the role of major rock-derived nutrients. In addition, catchment-scale mass and isotope budgets of Ba show that the measured riverine export of Ba is lower than the estimated delivery of Ba to the Earth surface through rock alteration. This indicates the existence of a missing Ba component, which we attribute to the formation of Ba-bearing particulate organics (possibly accumulating as soil organic matter or currently growing biomass within the catchments) and to organic-bound Ba exported as “unsampled” river particulate organic matter. Given our findings on the trace element Ba, we explore whether the river fluxes of most major rock-derived nutrients (K, Mg, Ca) might also be significantly affected by biological uptake or release. A first-order correction of river-derived silicate weathering fluxes from biological cycling shows that the carbon dioxide (CO2) consumption by silicate weathering at the mouth of the Amazon could be several times higher than the previously reported value of 13 × 109 mol CO2 yr−1 (Gaillardet et al., 1997). Overall, our study clearly shows that the chemical and isotope compositions of rivers in the Amazon – and most likely in other large river basins – bear a biological imprint, thereby challenging common assumptions made in weathering studies.