Eutrophication in freshwaters is often caused by riverine nutrient loads, particularly bioavailable forms of phosphorus (P). Phosphorus concentrations and forms in large rivers are often spatially and temporally dynamic due to external inputs and internal transformations. For many large watersheds, eutrophication management is impaired by a poor understanding of how total P is partitioned and transformed as it is transported downstream in the largest rivers. For example, harmful cyanobacteria blooms in Lake Erie are primarily caused by bioavailable P exports from the Maumee River watershed; yet, we have a poor understanding of how P cycling in this river shapes P exports to Lake Erie. To better understand these processes, we sampled major total P fractions, including soluble reactive phosphorus (SRP), at six sites along 204 km of the Maumee River between May and September during two years. We also measured (1) P species at the mouth of two major tributaries, the Auglaize and Tiffin Rivers, monthly and (2) net P uptake and release by seston and benthic sediment (during June and August) at all sites. We observed a downstream decline in SRP concentrations associated with an increase in chlorophyll-a concentrations. Yet, seston P uptake and release did not have clear longitudinal patterns, instead seston was a P sink in all months except for June upstream of the Auglaize confluence and September downstream of the Auglaize confluence. In June and August, benthic sediment switched from a P sink upstream of the Auglaize confluence to a P source downstream. Our results highlight a clear separation of P species and transformation processes in the Maumee River between sections upstream and downstream of the Auglaize River. This work helps to elucidate the processes contributing to spatial and temporal patterns in total P species in the Maumee River and delivery to Lake Erie.