Orchids are highly dependent on their mycorrhizal fungal partners for nutrient supply, especially during early developmental stages. In addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly N-enriched. We know almost nothing about the N form preferentially transferred to the plant or about the key molecular determinants required for N uptake and transfer. We identified, in the genome of the orchid mycorrhizal fungus Tulasnella calospora, two functional ammonium transporters and several amino acid transporters but found no evidence of a nitrate assimilation system, in agreement with the N preference of the free-living mycelium grown on different N sources. Differential expression in symbiosis of a repertoire of fungal and plant genes involved in the transport and metabolism of N compounds suggested that organic N may be the main form transferred to the orchid host and that ammonium is taken up by the intracellular fungus from the apoplatic symbiotic interface. This is the first study addressing the genetic determinants of N uptake and transport in orchid mycorrhizas, and provides a model for nutrient exchanges at the symbiotic interface, which may guide future experiments.