The Fe-depositing microorganism Gallionella ferruginea was first described in 1836 based on its association with Fe-rich environments and its distinctive morphology. Since then, this morphology has been widely used to identify G. ferruginea. Researchers have isolated several Fe-oxidizing bacteria (FeOB) related to Gallionella; however, few isolates have produced organized extracellular biomineral structures, and of these, only one stalk former has a sequenced 16S rRNA gene, listed as G. ferruginea in the GenBank database. Here we report the isolation and characterization of a novel stalk-forming Fe-oxidizing bacterium, strain R-1, from a freshwater Fe seep. Despite a strong morphological similarity to G. ferruginea, this isolate has only 93.55% 16S rRNA gene sequence similarity with the previously determined sequence. R-1 only grows on Fe(II) substrates, at pH 5.6 to 7.0 and from 10°C to 35°C, with a doubling time of ∼15 h at pH 6.3 and 22°C. It is a Betaproteobacterium, most closely related to uncultured bacteria from microaerobic Fe(II)-rich groundwater springs. The most closely related isolates are Sideroxydans spp. (94.05–94.42% sequence similarity), FeOB that are not known to produce morphologically distinct minerals. To our knowledge, this is the first reported stalk-forming freshwater FeOB isolate distinct from Gallionella.