Huge amounts of pollen can accumulate at the air–water interface during the floral bloom of wind-pollinated trees. We hypothesized that such pollen rains act as subsidy pulses, promoting the development of a neustonic microbial food web and the transfer of pollen organic carbon to aquatic consumers. During a pine pollen rain event on an oxbow lake, microorganism concentrations were by far higher in the neuston, where pollen grains accumulated, than in the seston. Zooplankton species were also unevenly distributed in the two compartments. Bulk isotope and isotopes of fatty acid analyses revealed trophic partitioning among these zooplankton species, with some of the taxa foraging specifically on neuston, where they benefit from pollen-derived carbon. Microorganisms were identified as a key element in the trophic upgrading of pollen food quality and in the transfer of pollen carbon to metazoan consumers. Pollen rains may thus contribute, as an allochthonous food pulse, to aquatic production at specific seasons, but they may also act as a structuring factor in lake habitats.