Potassium (K +) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K + channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K + conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K +-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K + contents at standard K + only. In such conditions, plants inoculated with these transgenic lines were impaired in K + nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K + available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K + transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K + nutrition of the host plant.