There is growing acknowledgement of the interaction between animals and the river bed on which they live and the implications of biological activity for geomorphic processes. It has been observed that signal crayfish (Pacifastacus leniusculus) disturb gravel substrates, potentially promoting sediment transport and impacting ecological communities. However, the mechanisms involved and the extent of their impact remain poorly understood, especially in relation to other processes that affect grain mobility in gravel-bed rivers. A series of flume experiments, using loose and water-worked gravel beds of narrowly graded grain sizes that were exposed to 6 h of crayfish activity under low-velocity flows, showed a substantial increase in the number of grains entrained by subsequent higher-velocity flows when compared with control runs in which crayfish were never introduced. Crayfish alter the topography of their substrate by constructing pits and mounds, which affect grain protrusion. When walking and foraging, they also alter gravel fabric by reorienting and changing the friction angle of surface grains. In water-worked surfaces, this fabric rearrangement is shown to lead to a statistically significant, partial reversal of the structuring that had been achieved by antecedent flow. For these previously water-worked surfaces, the increase in entrainment arising from disturbance by crayfish was statistically significant, with grain transport nearly twice as great. This suggests that signal crayfish, an increasingly widespread invasive species in temperate latitudes beyond their native NW North America, have the potential to enhance coarse-grained bedload flux by altering the surface structure of gravel river beds and reducing the stability of surface grains. This study illustrates further the importance of acknowledging the impact of mobile organisms in conditioning the river bed when assessing sediment entrainment mechanics in the context of predicting bedload flux. Copyright © 2011 John Wiley & Sons, Ltd.