The influence of Quaternary climate oscillations on the diversification of the South American fauna is being increasingly explored. However, most of these studies have focused on taxa that are endemic to tropical environments, and relatively few have treated organisms restricted to subtropical biomes. Here we used an integrative phylogeographical framework to investigate the effects of these climate events on the ecological niche and genetic patterns of the subtropical orb-weaver spider Araneus omnicolor (Araneidae). We analyzed the mitochondrial (Cytochrome Oxidase I, COI) and nuclear (Internal Transcribed Subunit II, ITS2) DNA of 130 individuals throughout the species’ range, and generated distribution models in three different climate scenarios [present, Last Glacial Maximum (LGM), and Last Interglacial Maximum (LIG)]. Additionally, we used an Approximate Bayesian Computation (ABC) approach to compare possible demographic scenarios and select the hypothesis that better explains the genetic patterns of A. omnicolor. We obtained high haplotype diversity but low nucleotide variation among sequences. The population structure and demographic analyses showed discrepancies between markers, suggesting male-biased dispersal in the species. The time-calibrated COI phylogenetic inference showed a recent diversification of lineages (Middle/Late Pleistocene), while the paleoclimate modeling indicated niche stability since ~120 Kya. The ABC results agreed with the niche models, supporting a panmictic population as the most likely historical scenario for the species. These results indicate that A. omnicolor experienced no niche or population reductions during the Late Pleistocene, despite the intense landscape modifications that occurred in the subtropical region, and that other factors beside LGM and LIG climate oscillations might have contributed to the demographic history of this species. This pattern may be related to the high dispersal ability and wide environmental tolerance of A. omnicolor, highlighting the need for more phylogeographical studies with invertebrates and other generalist taxa, in order to understand the effects of Quaternary climate changes on Neotropical biodiversity.