The salinization of freshwater ecosystems is emerging as a major ecological issue. Several anthropogenic causes of salinization (e.g. surface coal mining, hydro-fracking, road de-icing, irrigation of arid lands, etc.) are associated with biodiversity losses in freshwater ecosystems. Because insects tend to dominate freshwater ecology, it is important that we develop a better understanding of how and why different species respond to salinity matrices dominated by different major ions. This study builds upon previous work demonstrating that major ion toxicity to the mayfly Neocloeon triangulifer was apparently due to the ionic composition of water rather than specific conductance. Synthetic waters with low Ca:Mg ratios and high SO4:Na ratios produced toxicity, whereas waters with higher Ca:Mg ratios and lower SO4:Na ratios were not toxic to mayflies at comparable conductivities. Here we used a radiotracer approach to show that Mg did not competitively exclude Ca uptake at environmentally realistic ratios in 4 aquatic insect species. We characterized SO4 uptake kinetics in 5 mayflies and assessed the influence of different ions on SO4 uptake. Dual label experiments show an inverse relationship between SO4 and Na transport rates as SO4 was held constant and Na was increased, suggesting that Na (and not Cl or HCO3) is antagonistic to SO4 transport. Based on this observation, we tested the hypothesis that increasing Na would protect against SO4 induced toxicity in a Na-dependent manner. Increasing Na from 0.7 to 10.9mM improved 96-h survivorship associated with 20.8mM SO4 from 44% to 73% in a concentration dependent manner. However, when Na reached 21.8mM, survivorship decreased to 16%, suggesting that other interactive effects of major ions caused toxicity under those conditions. Thus, the combination of elevated sulfate and low sodium commonly observed in streams affected by mountaintop coal mining has the potential to cause toxicity in sensitive aquatic insects. Overall, it is important that we develop a better understanding of major ion toxicity to effectively mitigate and protect freshwater biodiversity from salinization.