Pimelodella spelaea, new species, is described from a subterranean stream tributary to the São Bernardo River inside the São Bernardo Cave, in the São Domingos karst area, upper Tocantins River basin, central Brazil. The new species can be distinguished from its epigean (surface) congeners by derived troglomorphic features such as smaller eyes and fainter coloration, which are nevertheless less reduced than in the other known troglobitic (exclusively subterranean) species, Pimelodella kronei, from southeast Brazil. Ecological data such as habitat characteristics, population data based on mark-recapture techniques, and distributions of frequencies of standard length, weight, and condition factor, were obtained during the dry season of 2000 (May to September). A relatively high population density (about one individual per m⁻²) was recorded for the accessible habitat, which is probably much lower in the nonaccessible, phreatic area of distribution. The low condition factor recorded for P. spelaea, which tended to decrease along the study period, indicates a regime of severe food limitation, intensifying along the dry season. An allopatric model of differentiation is hypothesized, either because of topographic isolation or because of local extinction of epigean relatives, or both factors combined. The conservation status of the new species is also discussed.