Many freshwater ecosystems are becoming saltier and/or warmer, but our understanding of how these factors interact and affect the physiology and life history outcomes of most aquatic species remain unknown. We hypothesize that temperature modulates ion transport rates. Since ion transport is energetically expensive, increases in salinity and/or temperature may influence ion flux rates and ultimately, organismal performance. Radiotracer (22Na+, 35SO4-2, and 45Ca2+) experiments with lab-reared mayflies (N. triangulifer) and other field-collected insects showed that increasing temperature generally increased ion transport rates. For example, increasing temperature from 15°C to 25°C, increased 22Na+ uptake rates by two-fold (p < 0.0001) and 35SO4-2 uptake rates by four-fold (p < 0.0001) in Hydropsyche sp.. Smaller changes in 22Na+ and 35SO4-2 uptake rates were observed in Isonychia sp. and Maccaffertium sp., suggesting species-specific differences in the thermal sensitivity of ion transport. We further explored the influence of SO4 challenge on mRNA expression of two SO4 transporter genes (putatively Na-dependent and Na-independent SO4 transporters in the SO4 permease family). Expression of the Na-dependent SO4 transporter was unaffected, whereas the expression of the Na-independent SO4 transporter was increased 4.5-fold at the highest salinity (1300 mg/L SO4) (p < 0.05), suggesting an efflux function. Finally, we demonstrated that the toxicity of SO4 was influenced by temperature profoundly in a 96-hour bioassay. Under the saltiest conditions (1500 mg/L SO4), mayfly survival was 78% at 15°C, but only 44% at 25°C (p < 0.0036). Conceivably, the energetic cost of osmoregulation in warmer, saltier environments may cause significant major ion toxicity in certain species of freshwater insects.