To determine the effects of anthropogenic pressures on the ecology of rivers in the Segura Watershed, we examined stream physicochemistry and the macroinvertebrate community in relation to land use, flow regulation by reservoirs, and the Tajo-Segura Inter-basin Transfer. The inter-basin transfer is thought to be the most important stressor because it affects the flow regime as well as water quality and quantity in an arid, typically water-stressed environment. The concentrations of major ions were strongly impacted by agricultural and urban land uses and the inter-basin transfer. There was a significant increase in conductivity and the concentrations of SO42− and other major ions downstream of the Tajo-Segura Transfer. The macroinvertebrate community was less diverse and composed of more pollution-tolerant taxa in areas affected by flow regulation and the inter-basin transfer. The rivers of study are at risk for salinization because of inputs from land use and the Tajo-Segura Transfer. Therefore, major ion and nutrient concentrations should be monitored. Currently, there is no limit specified for SO42− concentration in relation to aquatic ecosystem health in Spain. Measures are needed to reduce the effects of contamination and flow regulation to protect the aquatic ecosystem within the Segura Watershed. Copyright © 2012 John Wiley & Sons, Ltd.