Stable isotope abundances (δ18O and δ2H) in regional aquifers can provide important paleoclimate information. However, identifying paleoclimate signals can be complicated by cross-formational mixing and, potentially, by isotopic diffusion between aquifers and confining units. We examine controls on δ18O and δ2H distributions in the Wilcox aquifer of the northern Gulf Coastal Plain (USA). We sampled groundwater for δ18O, δ2H, Cl−, and 36Cl along a ~300 km downgradient transect. We developed a simplified, 1D numerical model of groundwater flow and 18O transport to assess the possible importance of isotopic diffusion between the aquifer and its confining units. Along the inferred flowpath, δ18O and δ2H values were depleted by as much as 1.3 and 8.2‰, respectively, as the Wilcox aquifer transitioned from unconfined to confined. However, they then gradually rose farther downgradient by up to 1.1 and 8.6‰. Chlorine-36 analyses and 14C analyses (from other studies) indicate that groundwater ages range from ~103 yr to ~8 × 105 yr. Modeling results indicate that the effect of diffusion on isotopic abundances is limited, whereas Cl− data indicate that cross-formational flow is limited. Therefore, we posit that confined groundwater in our study reflects a Pleistocene paleorecharge signal.