A wide variety of different cell types have been shown to respond to nanofabricated growth surfaces via the process of contact guidance, however little is known about the intracellular mechanisms that control these events. In the present study we have identified the multi-functional signalling adaptor protein, RACK1, as a novel negative regulator of contact guidance on custom-engineered nanometric grooves. We found that over-expression of RACK1 in human breast cancer cells leads to a pro-adherent morphology characterised by the formation of stress fibres and focal adhesions. Enforced expression of RACK1 also limits the response of cells to contact guidance on nanometric grooves. In contrast, ablation of RACK1 protein with specific anti-sense oligonucleotides led to a dramatic enhancement of bi-directional extension of cells on nanometrically deep grooved surfaces, with a corresponding loss of focal adhesions and stress fibres. RACK1 therefore exerts a tonic inhibitory effect on cell contact guidance, while positively promoting an adhesive phenotype. This is the first example of an intracellular signalling molecule involved in the regulation of cell contact guidance on nanometric growth surfaces.