Elemental hyperaccumulation protects plants from many aboveground herbivores. Little is known about effects of hyperaccumulation on belowground herbivores or their ecological interactions. To examine effects of plant selenium (Se) hyperaccumulation on nematode root herbivory, we investigated spatial distribution and speciation of Se in hyperaccumulator roots using X-ray microprobe analysis, and effects of root Se concentration on root-associated nematode communities. Perennial hyperaccumulators Stanleya pinnata and Astragalus bisulcatus, collected from a natural seleniferous grassland contained 100–1500 mg Se kg⁻¹ root dry weight (DW). Selenium was concentrated in the cortex and epidermis of hyperaccumulator roots, with lower levels in the stele. The accumulated Se consisted of organic (C-Se-C) compounds, indistinguishable from methyl-selenocysteine. The field-collected roots yielded 5–400 nematodes g⁻¹ DW in Baermann funnel extraction, with no correlation between root Se concentration and nematode densities. Even roots containing > 1000 mg Se kg⁻¹ DW yielded herbivorous nematodes. However, greenhouse-grown S. pinnata plants treated with Se had fewer total nematodes than those without Se. Thus, while root Se hyperaccumulation may protect plants from non-specialist herbivorous nematodes, Se-resistant nematode taxa appear to associate with hyperaccumulators in seleniferous habitats, and may utilize high-Se hyperaccumulator roots as food source. These findings give new insight into the ecological implications of plant Se (hyper)accumulation.